Recombinant, active caspases and uses thereof

ABSTRACT

Rev-caspases comprising a primary product in which the small subunit is N-terminal to the large subunit are provided. Rev-caspases are used for screening and identifying caspase inhibitors and enhancers. Rev-caspase genes can be delivered to cells for gene therapy.

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application is a divisional of U.S. application Ser. No. 09/227,721, filed Jan. 8, 1999; which application claims the benefit of priority from U.S. Provisional Application Serial No. 60/070,987, filed Jan. 9, 1998, now abandoned; all prior applications are hereby incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates generally to regulating apoptosis, and more particularly to the novel aspartate-specific cysteine proteases known as caspases, their coding regions, mutant forms thereof, and their use in screening assays and as pharmaceutical compositions for the controlled death of targeted cells to treat human disease.

BACKGROUND OF THE INVENTION

[0003] Tissue homeostasis is maintained by the process of apoptosis—that is, the normal physiological process of programmed cell death. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.

[0004] Since apoptosis functions in maintaining tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes that occurs with many autoimmune diseases. Inappropriate loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can alter the natural progression of many of these diseases.

[0005] Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates. The pathway, itself, is a cascade of proteolytic events analogous to that of the blood coagulation cascade.

[0006] Several gene families and products that modulate the apoptotic process have now been identified. One family is the aspartate-specific cysteine proteases (“caspases”). The caspase Ced-3, identified in C. elegans, is required for programmed cell death during development of the roundworm C. elegans. Ced-3 homologues as well as other caspases have been characterized. The human caspase family includes, for example, human ICE (interleukin-1-β converting enzyme) (caspase-1), ICE_(re1)III (caspase-4), ICE_(re1)III (caspase-5), Mch5 (caspase-8), Mch4 (caspase-10), ICE-LAP6 (caspase-9), Mch2 (caspase-6), CPP32 (caspase-3), ICE-LAP3 (casepase-7), ICH-1 (caspase-2), Caspase 11-14, and others.

[0007] The caspases share many features. In this regard, caspases are cysteine proteases (named for a cysteine residue in the active site) that cleave substrates at Asp-X bonds. Furthermore, the primary caspase product is a zymogen that requires proteolytic cleavage at specific internal aspartate residues for activation. The primary gene product is arranged such that the N-terminal peptide (prodomain) precedes a large subunit domain, which precedes a small subunit domain. Cleavage of a caspase yields two subunits, a large (generally approximately 20 kD) and a small (generally approximately 10 kD) subunit that associate non-covalently: to form a heterodimer, and, in some caspases, an N-terminal peptide of varying length (see FIG. 1). The heterodimer may combine non-covalently to form a tetramer.

[0008] Caspase zymogens are themselves substrates for caspases. Inspection of the interdomain linkages in each zymogen reveals target sites (i.e. protease sites) that indicate a hierarchical relationship of caspase activation. By analyzing such pathways, it has been demonstrated that caspases are required for apoptosis to occur. Moreover, caspases appear to be necessary for the accurate and limited proteolytic events which are the hallmark of classic apoptosis (see Salvesen and Dixit, Cell, 91: 443-446, 1997). However, when overexpressed in mammalian cells, the short prodomain caspases-3 and -6 cells are unable to undergo autocatalytic processing/activation and do not induce apoptosis. Thus, no cellular model system has been developed in which to test inhibitors of these caspases nor is gene delivery of a caspase commonplace.

[0009] Therefore, there exists a need in the art for methods of assaying compounds for their ability to affect caspase activity as well as for methods of regulating caspases in order to treat diseases and syndromes. The present invention provides recombinant caspase constructs that are active in cells, allowing the regulation of apoptosis for the treatment of pathology as well as providing methods and compositions for assaying compounds for caspase inhibitory and, thus, anti-apoptotic effects, while further providing other related advantages.

SUMMARY OF THE INVENTION

[0010] The present invention generally provides rev-caspases. In one aspect, the invention provides an isolated nucleic acid molecule encoding a rev-caspase. In certain embodiments, the rev-caspase is selected from the group consisting of rev-caspase-1, rev-caspase-2, rev-caspase-3, rev-caspase-4, rev-caspase-5, rev-caspase-6, rev-caspase-7, rev-caspase-8, rev-caspase-9, rev-caspase-10, rev-caspase-1 1, rev-caspase-12, rev-caspase-13, and rev-caspase-14. In other preferred embodiments, the rev-caspase is a human rev-caspase. Nucleic acid and amino acid sequences of rev-caspases are provided. The invention also provides rev-caspase proteins.

[0011] In another aspect, an expression vector comprising the nucleic acid molecule encoding rev-caspase is provided, wherein the sequence encoding rev-caspase is operatively linked to a promoter. In certain embodiments, the promoter is inducible, such as HIV LTR. Host cells transfected with the expression vectors are also provided.

[0012] In the present invention, methods of identifying an inhibitor or enhancer of caspase processing activity are provided, comprising: (a) contacting a sample containing an in vitro translated rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

[0013] In other aspects, methods are provided for identifying an inhibitor or enhancer of caspase processing activity, comprising: (a) contacting a cell transfected with the vector expressing rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

[0014] Methods are also provided for identifying an inhibitor or enhancer of caspase-mediated apoptosis, comprising: (a) contacting a cell transfected with the vector expressing rev-caspase with a candidate inhibitor or candidate enhancer or with a reference compound; and (b) detecting cell viability, wherein viability of cells contacted with a candidate is increased in the presence of an inhibitor and is decreased in the presence of an enhancer compared to cells contacted with a reference compound.

[0015] In other aspects, gene delivery vehicles, comprising the nucleic acid molecule encoding a rev-caspase are provided, wherein the rev-caspase sequence is operatively linked to a promoter. In preferred embodiments, the gene delivery vehicle is a retrovirus or adenovirus or the nucleic acid molecule is associated with a polycation. The gene delivery vehicle may further comprise a ligand that binds a cell surface receptor.

[0016] The invention also provides methods of treating cancer or autoimmune diseases, comprising administering to a patient the gene delivery vehicles disclosed herein.

[0017] These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, the various references set forth below that describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1A is a schematic representation of the processing and folding of human caspase-3 into the mature zymogen represented by bar and ribbon diagrams and is representative of other caspases. α helices are shown as spirals and the β strands are represented by arrows. The N- and the C-termini of the LS are labeled N-LS and C-LS, respectively, and the termini of the SS are similarly labeled N-SS and FIGS. 1B and C are schematic representations of rev-caspase-3 and -6, respectively. In both FIG. 1B and C the N-terminus of the SS and the C-terminus of the LS are labeled as in FIG. 1A and the linker region between the C-SS and the N-LS which includes the caspase-3 or -6 prodomain is represented by a thin line. Solid arrows indicate the cleavage sites (DEVDG (SEQ ID NO:38), Asp9 and Asp28-for rev-caspase-3 and VEIDA (SEQ ID NO:116), and Asp23-for rev-caspase-6) within the linker region. The hatched boxes represent a 15 residue-long T7-tag on the N-termini of the wild-type and the rev-caspases. All aspartate processing sites are indicated on the bar diagrams. FIG. 1B further depicts a schematic representation of the spontaneous folding of rev-caspase-3 into the mature zymogen represented a ribbon diagram. The ribbon diagram of rev-caspase-3 is based on the published crystal structure of caspase-3.

[0019]FIG. 2A is a scanned image of an autoradiogram representing SDS-PAGE analysis of rev-caspase autoprocessing. Caspase-3 and -6 or their rev-versions (Rev) including active site Cys to Ala mutants (Rev C/A) in pRSC-lacZ constructs were in vitro translated in the presence of ³⁵S-methionine. The translation products were then analyzed by SDS-PAGE and autoradiography. The LS and the SS are indicated.

[0020]FIGS. 2B and C are scanned images of autoradiograms representing SDS-PAGE analysis of the autoprocessing of rev-caspase-3 and -6, respectively in the presence of varying levels of selected caspase inhibitors. Rev-caspase-3 (FIG. 2B) or rev-caspase-6 (FIG. 2C) were in vitro translated in the presence of increasing concentrations of DEVD-CHO (0.04 μM); (SEQ ID NO:52), or zVAD-fmk (0-5 μM). The translation products were then analyzed as in FIG. 2A. WT, wild-type.

[0021]FIGS. 3A and B are scanned images representing the SDS-PAGE analysis of the ability of rev-caspase-3 (imaged by western blot) and -6 (imaged by autoradiogram) to cleave PARP and lamin, respectively. In FIG. 3A purified human PARP was incubated with buffer (lane 1) or BL-21 bacterial extracts prepared from bacteria transformed with caspase-3 (lane 2), rev-caspase-3 (lane 3), caspase-6 (lane 4), rev-caspase-6 (lane 5) constructs or empty pET28a vector (lane 6) for 2 h at 37° C. The reaction products were then analyzed by SDS-PAGE and Western blotting with anti-human PARP antibody. In FIG. 3B a cDNA encoding the C-terminus of lamin A (amino acids) which contain the caspase-6 cleavage site (VEIDA); (SEQ ID NO:116), was amplified by PCR and in vitro translated in the presence of ³⁵S-methionine. The labeled product was incubated with buffer (lane 1) or the BL-21 bacterial extracts listed above for 2 h at 37° C., and then analyzed by SDS-PAGE and autoradiography. The cleavage products are indicated to the right.

[0022]FIGS. 4A and B are bar diagrams representing the ability of rev-caspase-3 and -6 to induce apoptosis in MCF-7 cells. MCF-7 cells were transiently transfected with either rev-caspase-3 (FIG. 4A), or rev-caspase-6 (FIG. 4B) expression constructs in combination with 4-fold of CrmA, p35 or Bcl-2 expression constructs, or 20 μM zVAD-fmk. Cells transfected with an empty vector or the wild-type caspase-3 or -6 were used as controls.

[0023]FIG. 5A is a scanned image of an autoradiogram representing SDS-PAGE analysis of the enzymatic activity of uncleavable rev-caspase-3. Uncleavable rev-caspase-3 was in vitro translated in the absence or the presence of increasing concentrations of DEVD-CHO (SEQ ID NO:52),. The translation product contains a cleavable 35 residues-long His6-T7-tag at its N-terminus. The active site mutant rev-caspase-3 (Rev C/A) was used as a control. The p32 cleavage product without the His6-T7-tag is indicated to the right.

[0024]FIG. 5B is a plot of an activity assay of bacterially expressed uncleavable rev-caspase-3. The plot measures the ability of the uncleavable rev-caspase-3 to cleave the DEVD-AMC (SEQ ID NO:52), substrate. Rev, rev-caspase-3; Rev-mod, uncleavable rev-caspase-3; Rev-C/A, rev-caspase-3 with an active site mutation.

[0025]FIG. 6 is a multiple amino acid sequence alignment of the relatively conserved regions of the caspases (SEQ ID NOs:54-115). In the bottom line, “c” refers to residues involved in catalysis, “b” refers to residues that bind the substrate-carboxylate of P1 Asp, “a” refers to residues adjacent to the substrate P2-P4 recognition responsible amino acids, “DX” indicates known and potential processing sites between the small and large subunits of the caspases. The roman numerals at the left of the figure indicate the caspase subfamilies: Ced-like (I), ICE-like (II), and the Nedd2/Ich-1-like (III). The asterisk represents the non-conservative substitution in the active site pentapeptide sequences of Mch4 (caspase-10), Mch5 (caspase-8), and Mch6 (caspase-9)

[0026]FIG. 7 depicts a nucleotide sequence of Rev-caspase-3 (SEQ ID NO:1).

[0027]FIG. 8 depicts a nucleotide sequence of uncleavable Rev-caspase-3 (SEQ ID NO:2).

[0028]FIG. 9 depicts a nucleotide sequence of Rev-caspase-6 (SEQ ID NO:3).

[0029]FIG. 10 depicts a schematic of some possible rev-caspases. I, intervening sequence; SS, small subunit; P, prodomain; LS, large subunit; X, linker.

[0030]FIGS. 11A and 11B depict a nucleotide (SEQ ID NOs:4 and 5) and predicted amino acid sequence of caspase-1 (SEQ ID NO:6).

[0031]FIGS. 12A and 12B depict a nucleotide (SEQ ID NOs:7 and 8) and predicted amino acid sequence of caspase-2 (SEQ ID NO:9).

[0032]FIGS. 13A and 13B depict a nucleotide (SEQ ID NOs:10 and 11) and predicted amino acid sequence of caspase-3 (SEQ ID NO:12).

[0033]FIGS. 14A and 14B depict a nucleotide (SEQ ID NOs:13 and 14) and predicted amino acid sequence of caspase-4 (SEQ ID NO:15).

[0034]FIGS. 15A and 15B depict a nucleotide (SEQ ID NOs:16 and 17) and predicted amino acid sequence of caspase-5 (SEQ ID NO:18).

[0035]FIGS. 16A and 16B depict a nucleotide (SEQ ID NOs:19 and 20) and predicted amino acid sequence of caspase-6 (SEQ ID NO:21).

[0036]FIGS. 17A and 17B depict a nucleotide (SEQ ID NOs:22 and 23) and predicted amino acid sequence of caspase-7 (SEQ ID NO:24).

[0037] FIGS. 18A-18C depict a nucleotide (SEQ ID NOs:25 and 26) and predicted amino acid sequence of caspase-8 (SEQ ID NO:27).

[0038]FIGS. 19A and 19B depict a nucleotide (SEQ ID NOs:28 and 29) and predicted amino acid sequence of caspase-9 (SEQ ID NO:30).

[0039]FIGS. 20A and 20B depict a nucleotide (SEQ ID NOs:31 and 32) and predicted amino acid sequence of caspase-10 (SEQ ID NO:33).

[0040]FIGS. 21A, 21B, and 21C depict predicted amino acid sequences of Rev-caspase-3 (A; SEQ ID NO:34), uncleavable rev-caspase-3 (B; SEQ ID NO:35), and rev-caspase-6 (C; SEQ ID NO:36).

DETAILED DESCRIPTION OF THE INVENTION

[0041] Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter.

[0042] As used herein, a caspase refers to a cysteine protease that specifically cleaves proteins after Asp residues. Caspases are initially expressed as zymogens, in which a large subunit is N-terminal to a small subunit. Caspases are generally activated by cleavage at internal Asp residues (FIG. 1A). These proteins have been identified in many eukaryotes, including C. elegans, Drosophila, mouse, and humans. Currently, there are at least 14 known caspase genes, named caspase-1 through caspase-14. Caspases are found in myriad organisms, including human, mouse, insect ( e.g., Drosophila), and other invertebrates (e.g., C. elegans). In Table 1, ten human caspases are listed along with their alternative names. The nucleotide and amino acid sequences of representative human caspase gene products are presented in SEQ ID NOs:4-33 and FIGS. 11-20. Caspase Alternative name Caspase-1 ICE Caspase-2 ICH-1 Caspase-3 CPP32, Yama, apopain Caspase-4 ICE_(rel)II; TX, ICH-2 Caspase-5 ICE_(rel)III; TY Caspase-6 Mch2 Caspase-7 Mch3, ICE-LAP3, CMH-1 Caspase-8 FLICE; MACH; Mch5 Caspase-9 ICE-LAP6; Mch6 Caspase-10 Mch4, FLICE-2

[0043] As used herein, “rev-caspase” refers to a cysteine protease that specifically cleaves proteins after Asp residues and is expressed as a zymogen, in which a small subunit is N-terminal to a large subunit.

[0044] Within the context of this invention, it should be understood that a caspase or rev-caspase includes wild-type protein sequences, as well as other variants (including alleles) of the native protein sequence. Briefly, such variants may result from natural polymorphisms or may be synthesized by recombinant methodology, and differ from wild-type protein by one or more amino acid substitutions, insertions, deletions, or the like. Typically, when engineered, amino acid substitutions will be conservative, i.e., substitution of amino acids within groups of polar, non-polar, aromatic, charged, etc. amino acids. In the region of homology to the native sequence, variants should preferably have at least 90% amino acid sequence identity, and within certain embodiments, greater than 92%, 95%, or 97% identity.

[0045] As will be appreciated by those skilled in the art, a nucleotide sequence encoding a caspase, rev-caspase or variant may differ from the known native sequences, due to codon degeneracies, nucleotide polymorphisms, or amino acid differences. In other embodiments, variants should preferably hybridize to the native nucleotide sequence at conditions of normal stringency, which is approximately 25-30° C. below Tm of the native duplex (e.g., 5×SSPE, 0.5% SDS, 5×Denhardt's solution, 50% formamide, at 42° C. or equivalent conditions; see generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). Low stringency hybridizations utilize conditions approximately 40° C. below Tm, and high stringency hybridizations utilize conditions approximately 10° C. below Tm. Variants preferably have at least 75% nucleotide identity to native sequence, preferably at least 80%, 85%, and most preferably at least 90% nucleotide identity.

[0046] An “isolated nucleic acid molecule” refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct, that has been separated from its source cell (including the chromosome it normally resides in) at least once in a substantially pure form. Nucleic acid molecules may be comprised of a wide variety of nucleotides, including DNA, RNA, nucleotide analogues, or some combination of these.

[0047] A. Caspase and Rev-caspase Genes and Gene Products

[0048] As noted above, the invention provides compositions relating to caspase and rev-caspase genes and gene products, and methods for the use of the genes and gene products. In particular, the invention provides rev-caspase constructs that are active when expressed in cells. Given the disclosure provided herein, a caspase gene can be isolated from a variety of cell types and engineered to produce a rev-caspase.

[0049] 1. Isolation of Caspase Genes

[0050] The present invention, as described herein, provides rev-caspase genes, which are constructed from caspase genes. Caspase genes may be isolated from either genomic DNA or preferably cDNA. Isolation of caspase genes from genomic DNA or cDNA typically can proceed by, first, generating an appropriate DNA library through techniques for constructing libraries that are known in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989) or purchased from commercial sources (e.g., Clontech, Palo Alto, Calif.). Briefly, cDNA libraries can be constructed in bacteriophage vectors (e.g. λZAPII), plasmids, or others, which are suitable for screening, while genomic DNA libraries can be constructed in chromosomal vectors, such as YACs (yeast artificial chromosomes), bacteriophage vectors, such as λEMBL3, λgt10, cosmids, or plasmids.

[0051] In one embodiment known caspase sequences may be utilized to design an oligonucleotide hybridization probe suitable for screening genomic or cDNA libraries. Preferably, such oligonucleotide probes are 20-30 bases in length. To facilitate hybridization detection, the oligonucleotide may be conveniently labeled, generally at the 5′ end, with a reporter molecule, such as a radionuclide, (e.g., ³²P), enzymatic label, protein label, fluorescent label, or biotin. Such libraries are then generally plated as phage or colonies, depending upon the vector used. Subsequently, a nitrocellulose or nylon membrane, to which the colonies or phage have been transferred, is probed to identify candidate clones which contain the caspase gene. Such candidates may be verified as containing caspase DNA by any of various means including, for example, DNA sequence analysis or hybridization with a second, non-overlapping probe.

[0052] Once a library is identified as containing a caspase gene, the gene can be isolated by amplification. Briefly, when using cDNA library DNA as a template amplification primers are designed based upon known caspase gene sequences (see GenBank Accession Nos. X65019 (caspase-1), U13021 (caspase-2), U13737 (caspase-3), U25804 (caspase-4), U28015 (caspase-5), U20536 (caspase-6), U37448 (caspase-7), U60520 (caspase-8), U56390 (caspase-9), U60519 (caspase-10) Y13089 (caspase- 11), Y13090 (caspase-12), AF078533 (caspase-13), AF092997 (caspase-14), and sequences provided herein). Amplification of cDNA libraries made from cells with high caspase activity is preferred. Primers for amplification are preferably derived from sequences in the 5′ and 3′ untranslated region in order to isolate a full-length cDNA. The primers preferably have a GC content of about 50% and contain restriction sites to facilitate cloning and do not have self-complementary sequences nor do they contain complementary sequences at their 3′ end (to prevent primer-dimer formation). The primers are annealed to cDNA or genomic DNA and sufficient amplification cycles are performed to yield a product readily visualized by gel electrophoresis and staining. The amplified fragment is purified and inserted into a vector, such as λgt10 or pBS(M13+), and propagated. Confirmation of the nature of the fragment is obtained by DNA sequence analysis or indirectly through amino acid sequencing of the encoded protein.

[0053] Other methods may also be used to obtain a caspase encoding nucleic acid molecule. For example, a nucleic acid molecule encoding caspase may be obtained from an expression library by screening with an antibody or antibodies reactive to caspase (see, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, 1987; Ausubel, et al. Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, NY, 1995).

[0054] Caspase genes from a variety of species may be isolated using the compositions provided herein. For closely related species, the human sequence or portion thereof may be utilized as a probe on a genomic or cDNA library. For example, a fragment of caspase that encompasses the catalytic site may be labeled and used as a probe on a library constructed from mouse, primate, rat, dog, or other vertebrate, warm-blooded or mammalian species. An initial hybridization at normal stringency may yield candidate clones or fragments. If no hybridization is initially observed, varying degrees of stringency may be used. (see Sambrook et al. supra, and other well-known sources for stringency conditions) While such probes may also be used to probe libraries from evolutionarily diverse species, such as Drosophila, hybridization conditions will likely be more relaxed.

[0055] While relaxed hybridization conditions using probes designed from human sequences may identify caspase genes of evolutionarily diverse species it may be more beneficial to attempt to directly isolate these genes from a library using methods which do not require the human sequence per se. These methods include, but are not limited to, amplification using primers derived from conserved areas, amplification using degenerate primers from various regions, antibody probing of expression libraries, and the like. For example, random-primed amplification (e.g., polymerase chain reaction) may be employed (see, e.g., Methods Enzymol. 254: 275, 1995; Trends Genet. 11: 242, 1995; Liang and Pardee, Science 257: 967, 1992; Welsh et al., Nucl. Acids Res. 20: 4965, 1992). In addition, variations of random-primed PCR may also be used, especially when a particular gene or gene family is desired. In such a method, one of the amplification primers is an “anchored oligo(dT) (oligo(dT)dN)” and the other primer is a degenerate primer based upon amino acid or nucleotide sequence of a related gene. A gene sequence is identified as a caspase by amino acid similarity and/or nucleic acid similarity. Generally, amino acid similarity is preferred. Candidate caspase genes are examined for enzyme activity by one of the functional assays described herein or other equivalent assays.

[0056] Variants of caspase and rev-caspase genes provided herein may be engineered from natural variants (e.g., polymorphisms, splice variants, mutants), synthesized or constructed. Many methods have been developed for generating mutants (see, generally, Sambrook et al., supra; Ausubel, et al., supra, and the discussion above). Briefly, preferred methods for generating a few nucleotide substitutions utilize an oligonucleotide that spans the base or bases to be mutated and contains the mutated base or bases. The oligonucleotide is hybridized to complementary single stranded nucleic acid and second strand synthesis is primed from the oligonucleotide. The double-stranded nucleic acid is prepared for transformation into host cells, typically E. coli, but alternatively, other prokaryotes, yeast or other eukaryotes. Standard screening and vector growth protocols are used to identify mutant sequences and obtain high yields.

[0057] Similarly, deletions and/or insertions of the caspase or rev-caspase genes may be constructed by any of a variety of known methods as discussed supra. For example, the gene can be digested with restriction enzymes and religated such that a sequence is deleted or religated with additional sequences such that an insertion or large substitution is made. Other means of generating variant sequences may be employed with methods known in the art, for example those described in Sambrook et al. (supra) and Ausubel et al. (supra). Verification of variant sequences is typically accomplished by restriction enzyme mapping, sequence analysis, or probe hybridization. Variants which catalyze Asp-specific cleavages are useful in the context of this invention.

[0058] B. Rev-Caspases

[0059] The caspases of the present invention are generated by rearranging the gene sequence of the caspase gene such that the nucleic acid sequence encoding the small subunit precedes (is 5′ to) the nucleic acid sequence encoding the large subunit. These rearranged caspases are called rev-caspases.

[0060] 1. Structure of Rev-caspases

[0061] The rev-caspases of the present invention comprise at least a portion of the small subunit and at least a portion of the large subunit. In preferred embodiments, the prodomain or a portion thereof (see FIGS. 1, 10) and/or an intervening sequence or a portion thereof (see FIG. 10) are also present in rev-caspase. In other preferred embodiments, a “linker” region is located between the small and large subunits.

[0062] The boundaries of the small subunit and large subunit are identified either experimentally by amino acid sequence analysis of the mature caspase or by inspection of structural homology (e.g., the conserved Asp-X cleavage site). For exemplary purposes, the Table below presents the boundaries of the prodomain (P), large subunit (LS), intervening sequence (I), and small subunit (SS) of human caspase-1 through -10. The nucleotide numbers refer to the nucleotides in SEQ ID NOs:4-33 and in FIGS. 11-20. Intervening Caspase Prodomain Large Subunit sequence Small Subunit Caspase-1 1-357 358-891  892-948 949-1212 Caspase-2 1-456 457-948  949-990 991-1305 Caspase-3 1-84  85-525 526-831  Caspase-4 1-240 241-810  811-867 868-1131 Caspase-5 1-363 364-933  934-1254 Caspase-6 1-69  70-537 538-579 580-879  Caspase-7 1-69  70-594 595-909  Caspase-8 1-681 682-1173 1174-1488  Caspase-9 1-390 391-945  946-990 991-1248 Caspase-10 1-657 658-1116 1117-1437 

[0063] As noted above, a portion of the large subunit and small subunit may be used in rev-caspase constructs. When designing rev-caspases that contain a portion of these subunits, the active site (e.g., QACXG, where X is Arg, Gln, or Gly); (SEQ ID NO:51), which is located near the C-terminus of the large subunit should not be deleted if protease activity is desired. Preferably, the 3-dimensional structure as determined by X-ray crystallography (see Mittl et al., J. Biol. Chem., 272: 6539-6547, 1997; Rotondu et al., Nat. Struct. Biol., 3: 619-625, 1996; Walker et al., Cell, 78: 343-352, 1994; Wilson et al., Nature, 370: 270-275, 1994) is maintained. For example, from the x-ray crystallographic structures of caspases, the amino acids, that are important in binding substrates have been identified. Likewise, substitutions of amino acids in the active site may be detrimental to maintaining activity. Although it is preferred that both subunits are derived from the same caspase, combinations of subunits from different caspases and/or from different species may be used.

[0064] The prodomain (sometimes called an N-terminal peptide) is generally not required for enzyme activity and is normally released in vivo. Rev-caspases of the present invention optionally have a prodomain or portion thereof. Similarly, the intervening sequence, which is present in certain caspases, is optional for inclusion in rev-caspases.

[0065] In certain embodiments, a linker region is engineered between the small and large subunits. A “linker region”, as used herein, refers to a peptide of from about 5 to about 50 amino acids. In preferred embodiments, the linker may contain a protease sensitive or cleavage site. Any site recognized by an intracellular protease may be used. In addition, multiple protease sensitive sites may be tandemly arranged in the same linker. Preferred protease sensitive sites are susceptible to cleavage by caspases or by viral proteases. Preferred caspase sensitive sites include, but are not limited to DXXDG (wherein X is any amino acid; SEQ ID NO:37); DEVDG (SEQ ID NO:38), IETDG (SEQ ID NO:39), YVADG (SEQ ID NO:40), YVHDG (SEQ ID NO:41), and WEHDG (SEQ ID NO:42). Furthermore, the Gly residue may be Ala or another small amino acid. The latter three sites are specifically cleaved by caspases-1, -4, and -5. Other sites specifically cleaved by only one or a few caspases are preferred in certain embodiments. Viral proteases cleavage sites include, but are not limited to, those recognized and cleaved by HIV protease, HCV (hepatitis C virus) protease, HBV (hepatitis B virus) protease, and rhinovirus protease.

[0066] 2. Construction of Rev-caspases

[0067] Rev-caspases may be constructed from caspase sequences by a variety of methods known in the art. A preferred method is amplification (e.g., polymerase chain reaction (PCR)) to selectively amplify the individual subunits and place these in cloning vectors such as pUC such as described in Example 1. Moreover, such PCR reactions can be performed in a variety of ways such that the primers used for amplification contain specific restriction endonuclease sites to facilitate insertion into a vector.

[0068] Further, a variety of other methodologies besides PCR may be used to attain the desired rearrangement. For example, one skilled in the art may employ isothermal methods to amplify the nucleotide sequence of interest, using existing restriction endonuclease sites present in the nucleotide sequence to excise and insert sequences, or by the introduction of distinct restriction endonuclease sites by site-directed mutagenesis followed by excision and insertion. These and other methods are described in Sambrook et al., supra; Ausubel, et al., supra. Briefly, one methodology is to generate single-stranded cDNA of the caspase of interest, followed by annealing a primer, which is complementary except for the desired alteration (e.g., a small insertion, deletion, or mutation such that a unique restriction site is created between the large and small subunits and/or at the 5′ and 3′ ends of both subunits). Bacterial cells are transformed and screened for those which contain desired construct. This construct is then digested to liberate the subunit sequences, which can then be purified and religated into the appropriate orientation.

[0069] As indicated above, rev-caspase genes may be manipulated to contain insertions, deletions or substitutions. Moreover, such variant rev-caspase genes useful in the context of this invention include those which facilitate Asp-specific cleavages indicative of caspase activity. Further, variants which are incapable of being cleaved into separate subunits are encompassed within the context of this invention, if those variants are able to facilitate Asp-specific cleavages by way of a cysteine-containing active site. By way of guidance, amino acids involved in catalysis, Asp recognition in substrate, and P2-P4 substrate recognition are provided in FIG. 6.

[0070] C. Vectors, Host Cells and Means of Expressing and Producing Protein

[0071] Caspase may be expressed in a variety of host organisms. In certain embodiments, caspase is produced in bacteria, such as E. coli, or mammalian cells (e.g., CHO and COS-7), for which many expression vectors have been developed and are available. Other suitable host organisms include other bacterial species, and eukaryotes, such as yeast (e.g., Saccharomyces cerevisiae), and insect cells (e.g., Sf9).

[0072] A DNA sequence encoding rev-caspase is introduced into an expression vector appropriate for the host. In certain embodiments, rev-caspase is inserted into a vector such that a fusion protein is produced. The rev-caspase sequence is derived as described herein. As discussed above, the sequence may contain alternative codons for each amino acid with multiple codons. The alternative codons can be chosen as “optimal” for the host species. Restriction sites are typically incorporated into the primer sequences and are chosen with regard to the cloning site of the vector. If necessary, translational initiation and termination codons can be engineered into the primer sequences.

[0073] At minimum, the vector must contain a promoter sequence. As used herein, a “promoter” refers to a nucleotide sequence that contains elements that direct the transcription of a linked gene. At minimum, a promoter contains an RNA polymerase binding site. More typically, in eukaryotes, promoter sequences contain binding sites for other transcriptional factors that control the rate and timing of gene expression. Such sites include TATA box, CAAT box, POU box, AP1 binding site, and the like. Promoter regions may also contain enhancer elements. When a promoter is linked to a gene so as to enable transcription of the gene, it is “operatively linked”.

[0074] Other regulatory sequences may be included. Such sequences include a transcription termination signal sequence, secretion signal sequence, origin of replication, selectable marker, and the like. The regulatory sequences are operationally associated with one another to allow transcription or translation.

[0075] The expression vectors used herein include a promoter designed for expression of the proteins in a host cell (e.g., bacterial). Suitable promoters are widely available and are well known in the art. Inducible or constitutive promoters are preferred. Such promoters for expression in bacteria include promoters from the T7 phage and other phages, such as T3, T5, and SP6, and the trp, lpp, and lac operons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tac and trc, may also be used. Promoters for expression in eukaryotic cells include the P10 or polyhedron gene promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784), MMTV LTR, CMV IE promoter, RSV LTR, SV40, metallothionein promoter (see, e.g., U.S. Pat. No. 4,870,009) and the like.

[0076] The promoter controlling transcription of rev-caspase may itself be controlled by a repressor. In some systems, the promoter can be derepressed by altering the physiological conditions of the cell, for example, by the addition of a molecule that competitively binds the repressor, or by altering the temperature of the growth media. Preferred repressor proteins include, but are not limited to the E. coli lacI repressor responsive to IPTG induction, the temperature sensitive λc1857 repressor, and the like. The E. coli lacI repressor is preferred.

[0077] In other preferred embodiments, the vector also includes a transcription terminator sequence. A “transcription terminator region” has either a sequence that provides a signal that terminates transcription by the polymerase that recognizes the selected promoter and/or a signal sequence for polyadenylation.

[0078] Preferably, the vector is capable of replication in the host cells. Thus, when the host cell is a bacterium, the vector preferably contains a bacterial origin of replication. Preferred bacterial origins of replication include the fl-ori and col E1 origins of replication, especially the ori derived from pUC plasmids. In yeast, ARS or CEN sequences can be used to assure replication. A well-used system in mammalian cells is SV40 ori.

[0079] The plasmids also preferably include at least one selectable marker that is functional in the host. A selectable marker gene includes any gene that confers a phenotype on the host that allows transformed cells to be identified and selectively grown. Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Amp^(r)), tetracycline resistance gene (Tc^(r)) and the kanamycin resistance gene (Kan^(r)). The kanamycin resistance gene is presently preferred. Suitable markers for eukaryotes usually require a complementary deficiency in the host (e.g., thymidine kinase (tk) in tk- hosts). However, drug markers are also available (e.g., G418 resistance and hygromycin resistance).

[0080] The sequence of nucleotides encoding rev-caspase may also include a secretion signal, whereby the resulting peptide is a precursor protein processed and secreted. The resulting processed protein may be recovered from the periplasmic space or the fermentation medium. Secretion signals suitable for use are widely available and are well known in the art (von Heijne, J. Mol. Biol. 184: 99-105, 1985). Prokaryotic and eukaryotic secretion signals that are functional in E. coli (or other host) may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: pelB (Lei et al., J. Bacteriol. 169: 4379, 1987), phoA, ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase.

[0081] One skilled in the art appreciates that there are a wide variety of suitable vectors for expression in bacterial cells and which are readily obtainable. Vectors such as the pET series (Novagen, Madison, Wis.), the tac and trc series (Pharmacia, Uppsala, Sweden), pTTQ18 (Amersham International plc, England), pACYC 177, pGEX series, and the like are suitable for expression of a rev-caspase. Baculovirus vectors, such as pBlueBac (see, e.g., U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego) may be used for expression in insect cells, such as Spodoptera frugiperda sf9 cells (see, U.S. Pat. No. 4,745,051). The choice of a bacterial host for the expression of a rev-caspase is dictated in part by the vector. Commercially available vectors are paired with suitable hosts.

[0082] A wide variety of suitable vectors for expression in eukaryotic cells are available. Such vectors include pCMVLacI, pXT1 (Stratagene Cloning Systems, La Jolla, Calif.); pCDNA series, pREP series, pEBVHis (Invitrogen, Carlsbad, Calif.). In certain embodiments, rev-caspase gene is cloned into a gene targeting vector, such as pMClneo, a pOG series vector (Stratagene Cloning Systems).

[0083] Rev-caspase is isolated by standard methods, such as affinity chromatography, size exclusion chromatography, metal ion chromatography, ionic exchange chromatography, HPLC, and other known protein isolation methods. (see generally Ausubel et al. supra; Sambrook et al. supra). An isolated purified protein gives a single band on SDS-PAGE when stained with Coomassie blue.

[0084] Rev-caspase may be expressed as a hexa-his fusion protein and isolated by metal-containing chromatography, such as nickel-coupled beads. Briefly, a sequence encoding His₆ is linked to a DNA sequence encoding a rev-caspase. Although the His₆ sequence can be positioned anywhere in the molecule, preferably it is linked at the 3′ end immediately preceding the termination codon. The fusion may be constructed by any of a variety of methods. A convenient method is amplification of the rev-caspase gene using a downstream primer that contains the codons for His₆.

[0085] Purified rev-caspase protein may be used in assays to screen for inhibitory drugs. These assays may be performed in vitro or in vivo and utilize any of the methods described herein or that are known in the art. The protein may also be crystallized and subjected to X-ray analysis to determine its 3-dimensional structure or used to raise antibodies.

[0086] D. Uses of Rev-caspase Gene and Gene Product

[0087] 1. Inhibitors and Enhancers of Caspase Activity

[0088] Candidate inhibitors and enhancers may be isolated or procured from a variety of sources, such as bacteria, fungi, plants, parasites, libraries of chemicals, peptides or peptide derivatives and the like. Inhibitors and enhancers may be also be rationally designed, based on the protein structure determined from X-ray crystallography (see, Mittl et al., J. Biol. Chem., 272: 6539-6547, 1997). In certain preferred embodiments, the inhibitor targets a specific caspase (e.g., caspase-3 and not any other caspases).

[0089] Without being held to a particular mechanism, the inhibitor may act by preventing processing of caspase or by preventing enzymatic activity, or by other mechanism. The inhibitor may act directly or indirectly. In preferred embodiments, inhibitors interfere in the processing of the caspase protein. In other preferred embodiments, the inhibitors are small molecules. In a most preferred embodiment, the inhibitors prevent apoptosis. Inhibitors should have a minimum of side effects and are preferably non-toxic. Inhibitors that can penetrate cells are preferred.

[0090] In addition, enhancers of caspase activity or expression are desirable in certain circumstances. At times, increasing apoptosis will have a therapeutic effect. For example, tumors or cells that mediate autoimmune diseases are appropriate cells for destruction. Enhancers may increase the rate or efficiency of caspase processing, increase transcription or translation, or act through other mechanisms. As is apparent to one skilled in the art, many of the guidelines presented above apply to the design of enhancers as well.

[0091] Screening assays for inhibitors and enhancers will vary according to the type of inhibitor or enhancer and the nature of the activity that is being affected. Assays may be performed in vitro or in vivo. In general, in vitro assays are designed to evaluate caspase protein processing or caspase enzymatic activity, and in vivo assays are designed to evaluate caspase protein processing, caspase enzymatic activity, apoptosis, or caspase cleavage of substrate. In any of the assays, a statistically significant increase or decrease compared to a proper control is indicative of enhancement or inhibition.

[0092] One in vitro assay can be performed by examining the effect of a candidate compound on processing of rev-caspase into two subunits. Briefly, a cleavable form of rev-caspase, that is a primary translation product, is obtained from an in vitro translation system. The cleavable form of rev-caspase is preferably constructed to be auto-cleaved, but can be constructed to be cleaved by other protease components present or added to the reaction. This primary product is contacted with or without or translated in the presence or absence of a candidate compound and assessed for appearance of the two subunits. to facilitate detection, typically, the primary product of rev-caspase is labeled during translation, cell viability, and the like. The two subunits may be readily detected by autoradiography after gel electrophoresis. One skilled in the art will recognize that other methods of labeling and detection may be used alternatively.

[0093] An alternative in vitro assay is designed to measure cleavage of a caspase substrate (e.g., Acetyl DEVD-aminomethyl coumarin (amc) (SEQ ID NO:52),, lamin, PRPP, and the like). Substrate turnover may be assayed using either cleavable or noncleavable rev-caspase. Briefly, in this method, rev-caspase is translated and allowed sufficient time to be processed, if a cleavable rev-caspase is being used. The caspase substrate along with the candidate compound is added to the reaction. Detection of cleaved substrate is performed by any one of a variety of standard methods. Generally, the substrate will be labeled and followed by an appropriate detection means.

[0094] Moreover, any known enzymatic analysis can be used to follow the inhibitory or enhancing ability of a candidate compound with regard to a rev-caspase of this invention. For example, one could express the rev-caspase of interest in a cell line be it bacterial, insect, mammalian or other, either in cleavable or noncleavable form and purify the rev-caspase. The purified rev-caspase could then be used in a variety of assays to follow its catalytic ability in the presence of candidate compounds, as noted above. Such methods of expressing and purifying recombinant proteins are known in the art and examples can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989 as well as in a number of other sources.

[0095] In vivo assays are typically performed in cells transfected either transiently or stably with an expression vector containing a rev-caspase gene, such as those described herein. These cells are used to measure rev-caspase processing, substrate turnover, or apoptosis in the presence or absence of a candidate compound. When assaying apoptosis, a variety of cell analyses may be used including, for example, dye staining and microscopy to examine nucleic acid fragmentation and porosity of the cells. Further, in vivo assaying for the ability of the transfected rev-caspase to cleave known substrates that are co-transfected or placed in the cell culture media in the presence of the candidate compound can be performed thereby allowing for the detection and determination of substrate turnover.

[0096] The assays briefly described herein may be used to identify an enhance or inhibitor that is specific for an individual caspase. In a preferred embodiment candidate compounds would be analyzed using a variety of rev-caspases (e.g., rev-caspase-1 through rev-caspase-14) to identify specific for individual caspases.

[0097] A variety of methodologies exist can be used to investigate the effect of a candidate compound. Such methodologies are those commonly used to analyze enzymatic reactions and include, for example, SDS-PAGE, spectroscopy, HPLC analysis, autoradiography, chemiluminescence, chromogenic reactions, and immunochemistry (e.g., blotting, precipitating, etc.).

[0098] Inhibitors and enhancers may be used in the context of this invention to exert control over the cell death process or cytokine activation (e.g., IL-1, which is activated by caspase-1). Thus, these inhibitors and enhancers will have utility in diseases characterized by either excessive or insufficient levels of apoptosis. Inhibitors of proteases have potential to treat the major neurodegenerative diseases: stroke, Parkinson's Disease, Alzheimer's Disease, and ALS. As well, caspase protease inhibitors may be used to inhibit apoptosis in the heart following myocardial infarction, in the kidney following acute ischemia, and in diseases of the liver. In other embodiments, inhibitors of caspase-1 can be used to inhibit the release of the pro-inflammatory IL-1β, and thus may provide therapeutic benefit in treating inflammation and/or autoimmune disorders. Enhancers of caspase activity may be used in contexts when apoptosis or cytokine activation are desired. For example, inducing or increasing apoptosis in cancer cells or aberrantly proliferating cells may be effected by delivery of a caspase enhancer.

[0099] The inhibitors and enhancers may be further coupled with a targeting moiety that binds a cell surface receptor specific to the cells. Administration of inhibitors or enhancers will generally follow established protocols. The compounds of the present invention may be administered either alone, or as a pharmaceutical composition. Briefly, pharmaceutical compositions of the present invention may comprise one or more of the inhibitors or enhancers as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like, carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and preservatives. In addition, pharmaceutical compositions of the present invention may also contain one or more additional active ingredients.

[0100] Compositions of the present invention may be formulated for the manner of administration indicated, including for example, for oral, nasal, venous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. Within other embodiments of the invention, the compositions described herein may be administered as part of a sustained release implant. Within yet other embodiments, compositions of the present invention may be formulized as a lyophilizate, utilizing appropriate excipients which provide stability as a lyophilizate, and subsequent to rehydration.

[0101] 2. Gene Therapy

[0102] As noted above, rev-caspases may be delivered to cells as part of gene delivery vehicles. In many diseases and syndromes, too little apoptosis is an important feature in their development. Treatment of many autoimmune diseases and tumors would benefit from increased apoptosis. One means to increase apoptosis is to provide target cells with caspase genes in an expressible form. This may be accomplished by delivery of DNA or cDNA capable of in vivo transcription of the rev-caspase. More specifically, in order to produce rev-caspases in vivo, a nucleic acid sequence coding for the rev-caspase is placed under the control of a eukaryotic promoter (e.g., a pol III promoter, CMV or SV40 promoter). Where it is desired to more specifically control transcription, the rev-caspase may be placed under the control of a tissue or cell specific promoter (e.g., to target cells in the liver), or an inducible promoter, such as metallothionein.

[0103] Many techniques for introduction of nucleic acids into cells are known. Such methods include retroviral vectors and subsequent retrovirus infection, adenoviral or adeno-associated viral vectors and subsequent infection, and complexes of nucleic acid with a condensing agent (e.g., poly-lysine). These complexes or viral vectors may be targeted to particular cell types by way of a ligand incorporated into the vehicle. Many ligands specific for tumor cells and other cells are well known in the art.

[0104] A wide variety of vectors may be utilized within the context of the present invention, including for example, plasmids, viruses, retrotransposons and cosmids. Representative examples include adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Yei et al., Gene Therapy 1: 192-200, 1994; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J Neurosci. 5(10):1287-1291, 1993), adeno-associated type 1 (“AAV-1”) or adeno-associated type 2 (“AAV-2”) vectors (see WO 95/13365; Flotte et al., PNAS 90(22):10613-10617, 1993), hepatitis delta vectors, live, attenuated delta viruses and herpes viral vectors (e.g., U.S. Pat. No. 5,288,641), as well as vectors which are disclosed within U.S. Pat. No. 5,166,320. Other representative vectors include retroviral vectors (e.g., EP 0 415 731; WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218.

[0105] Within certain aspects of the invention, nucleic acid molecules that encode the rev-caspase may be introduced into a host cell utilizing a vehicle, or by various physical methods. Representative examples of such methods include transformation using calcium phosphate precipitation (Dubensky et al., PNAS 81: 7529-7533, 1984), direct microinjection of such nucleic acid molecules into intact target cells (Acsadi et al., Nature 352: 815-818, 1991), and electroporation whereby cells suspended in a conducting solution are subjected to an intense electric field in order to transiently polarize the membrane, allowing entry of the nucleic acid molecules. Other procedures include the use of nucleic acid molecules linked to an inactive adenovirus (Cotton et al., PNAS 89: 6094, 1990), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1989), microprojectile bombardment (Williams et al., PNAS 88: 2726-2730, 1991), polyeation compounds such as polylysine, receptor specific ligands, liposomes entrapping the nucleic acid molecules, spheroplast fusion whereby E. coli containing the nucleic acid molecules are stripped of their outer cell walls and fused to animal cells using polyethylene glycol, viral transduction, (Cline et al., Pharmac. Ther. 29: 69, 1985; and Friedmann et al., Science 244: 1275, 1989), and DNA ligand (Wu et al, J. of Biol. Chem. 264: 16985-16987, 1989), as well as psoralen inactivated viruses such as Sendai or Adenovirus. In one embodiment, the rev-caspase construct is introduced into the host cell using a liposome.

[0106] As noted above, pharmaceutical compositions also are provided by this invention. These compositions may contain any of the above described inhibitors, enhancers, DNA molecules, vectors or host cells, along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.

[0107] In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes, including for example intraarticularly, intracranially, intradermally, intrahepatically, intramuscularly, intraocularly, intraperitoneally, intrathecally, intravenously, subcutaneously or even directly into a tumor. In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition. Pharmaceutical compositions are useful for both diagnostic or therapeutic purposes.

[0108] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease. Dosages may be determined most accurately during clinical trials. Patients may be monitored for therapeutic effectiveness by appropriate technology, including signs of clinical exacerbation, imaging and the like.

[0109] The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES Example 1 Generation of cDNAs Expressing Rev-caspase-3 and 6 Precursors

[0110] Generation of cDNAs encoding rev-caspase-3 and 6 precursors were generated by PCR. The large (LS) and small subunits (SS) of caspase-3 were amplified with the following primers using the caspase-3 cDNA as a template: LS-forward, ATGGAGAACACTGAAAACTCAG (SEQ ID NO:43); LS-reverse, GTCATCATCAACACCTCAGTCT (SEQ ID NO:44); SS-forward, GGATCCATGATTGAGACAGACAGTGG (SEQ ID NO:45); SS-reverse, ATCAACTTCATCGTGATAAAAATAGAGTTC (SEQ ID NO:46).

[0111] The PCR products were cloned separately into the Sma I site of pBluescript KS⁺. The small subunit was then excised from KS⁺-vector with Bam HI and inserted into the Bam HI site of the second KS⁺-vector which contains the large subunit. This places the small subunit in-frame 5′ to the large subunit. Rev-caspase-6 was amplified and cloned in the KS⁺-vector in a similar way. The following PCR primers were used with caspase-6-His6 cDNA as a template: LS-forward, ATGAGCTCGGCCTCGGGG (SEQ ID NO:47); LS-reverse, TTAATCTACTACATCCAAAGG (SEQ ID NO:48); SS-forward, GGATCCATGGTAGAAATAGATGCAGCCTCCGTTTAC (SEQ ID NO:49) SS-reverse, ATCAATTTCAACGTGGTGGTGGTGGTGGTGC (SEQ ID NO:50).

[0112] The resulting nucleotide sequences were such that the wild type subunit order was reversed thus creating a contiguous nucleotide sequence wherein the coding region for the small subunit preceded that of the large subunit (See FIG. 1). The engineered contiguous caspase-3 and 6 molecules (i.e., rev-caspase molecules) in which the SS was fused in frame N-terminal to the LS, and a cleavage site (DEVDG in the case of caspase-3; VEIDS in the case of caspase-6 (these internal cleavage sites were designed to be specific for the caspase in which it was introduced in order to investigate the autocatalytic activity of the particular caspase)) was introduced between the two subunits and is depicted in FIG. 1B and C.

[0113] To express the rev-caspases in bacteria, their cDNAs were excised with Bam HI/Xho I and subcloned into the bacterial expression vector pET28a (Novagen, Inc.) in-frame with the T7-tag of this vector.

Example 2 Expression of Rev-caspase in Mammlian Cells and Assay for Apoptosis

[0114] To express the rev-caspases in mammalian cells and assay their apoptotic activity, they were amplified with the T7-tag primer and the LS-reverse primers using the pET28a constructs as templates, and subcloned into the mammalian double expression vector pRSC-LacZ (MacFarlane et al., J. Biol. Chem., 272: 25417-25420, 1997; Tsang et al., Bio/Technology, 22: 68, 1997). This vector allows the expression of lacZ under the Rous Sarcoma virus promoter, and the test cDNA under the CMV promoter. To assay for apoptosis, MCF-7 or 293 cells were transfected, using the method commercially available as the Lipofect Amine method (Life Technologies, Inc.), with the pRSC-LacZ constructs in the presence or absence of different apoptosis-inhibitors. 30 h after transfection cells were stained with β-galactosidase and examined for morphological signs of apoptosis. The percentage of round blue apoptotic cells (mean±SD) were represented as a function of total blue cells under each condition (n≧3).

Example 3 In Vitro Translation of Caspases

[0115]³⁵S-labeled caspases (wild-type and rev-caspases) were obtained by in vitro translation in the presence of ³⁵S-methionine using a coupled transcription/translation system in rabbit reticulocyte lysate using TNT Kit (Promega) according to the manufacturer's recommendations. Unlike the wild-type caspase-3 and 6, FIG. 2A demonstrates that rev-caspase-3 and 6 were able to undergo autocatalytic processing in the in vitro translation reaction. Further, this processing was completely inhibited by mutation of the active site Cys of rev-caspase-3 and 6 (FIG. 2A, lanes 3 and 6) and by selected caspase inhibitors (See Example 4). Because the in vitro translated products are present at very low concentration in the reaction mixture, the observed cleavage must be attributed to an intramolecular processing within the caspase heterotetramer.

Example 4 Effects of Inhibitors on Rev-caspase-3 and 6 Activity

[0116] To test the effect of selected caspase inhibitors on the autocatalytic activity of rev-caspase-3 and 6, the rev-caspases were translated as in Example 3, but in the presence of varying amounts of inhibitors. As demonstrated by FIG. 2B, in the presence of increasing amounts of DEVD-CHO (SEQ ID NO:52), a decrease in the amount of cleavage products and a corresponding increase in the amount of the revcaspase-3 precursor was observed. This corresponded to nearly 50-90% inhibition of the autocatalytic activity of rev-caspase-3 at 40-400 nM concentration. However, the same concentrations of this inhibitor had little effect on the autocatalytic activity of rev-caspase-6 (FIG. 2C). This is consistent with earlier observations that caspase-6 is poorly inhibited by DEVD-CHO (SEQ ID NO:52); see Srinivasula et al., J. Biol. Chem., 271: 27099-27106, 1996. On the other hand, as is apparent from inspection of FIGS. 2B and 2C, z-VAD-fmk had nearly an equal inhibitory effect on rev-caspase-3 and 6 autocatalytic activity at the concentration used in this experiment. Nevertheless, nearly 10-fold more of z-VAD-fink than DEVD-CHO (SEQ ID NO:52) was required to obtain complete inhibition of caspase-3 activity. Similarly, baculovirus p35 had nearly an equal inhibitory effect on rev-caspase-3 and 6 autocatalytic activity (data not shown).

Example 5 Caspase-3 and 6 Specificity Retention of Bacterially Expressed Rev-caspase-3 and -6

[0117] At limited caspase concentrations, poly(ADP) ribose polymerase (PARP) is specifically cleaved by caspase-3 and 7 but not other caspases. Similarly, lamin is specifically cleaved by caspase-6 but not other caspases. To compare the activity of the wild-type and rev-caspase-3 and 6 towards PARP and lamin, rev-caspase-3 and 6 were expressed in bacteria and then incubated with the two substrates PARP and lamin. As shown in FIGS. 3A and B, the activity of the rev-caspases towards these two substrates were indistinguishable from their wild-type counterparts. Both caspase-3 variants (rev and WT), but not caspase-6 variants efficiently cleaved PARP. In contrast, both caspase-6 variants, but not caspase-3 variants efficiently cleaved lamin. These results demonstrate that the mature caspases generated from the rev and the wild type constructs have identical substrate specificity.

Example 6 Induction of Apoptosis in Mammalian Cells by Rev-caspase-3 and -6

[0118] To determine the apoptotic activity of rev-caspase-3 and 6 in vivo, the rev-caspases were expressed in human MCF-7 cells, transfected as explained above in Example 2. As evidenced by FIGS. 4A and B, unlike the wild type caspase-3 and 6, the rev-caspases potently induced apoptosis in nearly 90% of the transfected cells. Overexpression of Bc1-2 or CrmA, which protect against different forms of apoptosis, did not significantly reduce their apoptotic activity. Nevertheless, overexpression of the baculovirus p35, which inhibits the activity of most caspases, partially protected against their apoptotic activity. Also, incubation of the transfected cells in the presence of 100 μM z-VAD-fmk, dramatically reduced their apoptotic activity to nearly 30%. These data demonstrate directly that the activity of caspase-3 and 6 are downstream of the CrmA and Bcl-2 block in the apoptotic cascade, and can only be inhibited by high concentration of the pancaspase-inhibitor z-VAD-fmk.

Example 7 Activity of Noncleavable Rev-caspase-3

[0119] To demonstrate that the rev-caspase molecules are inherently active and do not require separation of the two subunits and that the two subunits are derived from the same contiguous molecule, the DEVD (SEQ ID NO:52) site was removed and Asp9 and 28, that are present between the two subunits of rev-caspase-3, were mutated (see FIG. 2A). However, to follow the activity of this molecule a cleavable 35 residue long His6-T7-tag N-terminal to the IETD (SEQ ID NO:53) site was introduced (see FIG. 1B). FIG. 5 demonstrates that upon in vitro translation of this molecule, as described in Example 3 above, there was no evidence of cleavage between the two subunits. Nevertheless, the translated molecule was active as evident from its ability to cleave its T7-tag to form the p32 species (FIG. 5). In the presence of 400 nM DEVD-CHO (SEQ ID NO:52), processing of the T7-tag was inhibited and only the full length p34 species can be seen. Furthermore, expression of this molecule into MCF-7 cells potently induced apoptosis in these cells. These data demonstrate that when the two subunits of a caspase are rearranged in the reverse order, it is not necessary to separate them from each other to generate an active caspase. Thus by mimicking the mature caspase structure, it is possible to design a contiguous active caspase molecules.

[0120] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

1 116 1 873 DNA Artificial Sequence Rev-Caspase-3 contructed from human caspase -3 1 atgattgaga cagacagtgg tgttgatgat gacatggcgt gtcataaaat accagtggag 60 gccgacttct tgtatgcata ctccacagca cctggttatt attcttggcg aaattcaaag 120 gatggctcct ggttcatcca gtcgctttgt gccatgctga aacagtatgc cgacaagctt 180 gaatttatgc acattcttac ccgggttaac cgaaaggtgg caacagaatt tgagtccttt 240 tcctttgacg ctacttttca tgcaaagaaa cagattccat gtattgtttc catgctcaca 300 aaagaactct atttttatca cgatgaagtt gatgggggat cccccatgga gaacactgaa 360 aactcagtgg attcaaaatc cattaaaaat ttggaaccaa agatcataca tggaagcgaa 420 tcaatggact ctggaatatc cctggacaac agttataaaa tggattatcc tgagatgggt 480 ttatgtataa taattaataa taagaatttt cataagagca ctggaatgac atctcggtct 540 ggtacagatg tcgatgcagc aaacctcagg gaaacattca gaaacttgaa atatgaagtc 600 aggaataaaa atgatcttac acgtgaagaa attgtggaat tgatgcgtga tgtttctaaa 660 gaagatcaca gcaaaaggag cagttttgtt tgtgtgcttc tgagccatgg tgaagaagga 720 ataatttttg gaacaaatgg acctgttgac ctgaaaaaaa taacaaactt tttcagaggg 780 gatcgttgta gaagtctaac tggaaaaccc aaacttttca ttattcaggc ctgccgtggt 840 acagaactgg actgtggcat tgagacagac tga 873 2 858 DNA Artificial Sequence Uncleavalbe Rev-Caspase-3 contructed from human caspase -3 2 atgattgaga cagacagtgg tgttgatgat gacatggcgt gtcataaaat accagtggag 60 gccgacttct tgtatgcata ctccacagca cctggttatt attcttggcg aaattcaaag 120 gatggctcct ggttcatcca gtcgctttgt gccatgctga aacagtatgc cgacaagctt 180 gaatttatgc acattcttac ccgggttaac cgaaaggtgg caacagaatt tgagtccttt 240 tcctttgacg ctacttttca tgcaaagaaa cagattccat gtattgtttc catgctcaca 300 aaagaactct atttttatca cggatccccc atggagaaca ctgaaaactc agtggcttca 360 aaatccatta aaaatttgga accaaagatc atacatggaa gcgaatcaat ggcctctgga 420 atatccctgg acaacagtta taaaatggat tatcctgaga tgggtttatg tataataatt 480 aataataaga attttcataa gagcactgga atgacatctc ggtctggtac agatgtcgat 540 gcagcaaacc tcagggaaac attcagaaac ttgaaatatg aagtcaggaa taaaaatgat 600 cttacacgtg aagaaattgt ggaattgatg cgtgatgttt ctaaagaaga tcacagcaaa 660 aggagcagtt ttgtttgtgt gcttctgagc catggtgaag aaggaataat ttttggaaca 720 aatggacctg ttgacctgaa aaaaataaca aactttttca gaggggatcg ttgtagaagt 780 ctaactggaa aacccaaact tttcattatt caggcctgcc gtggtacaga actggactgt 840 ggcattgaga cagactga 858 3 903 DNA Artificial Sequence Rev-Caspase-6 contructed from human caspase -3 3 atggtagaaa tagatgcagc ctccgtttac acgctgcctg ctggagctga cttcctcatg 60 tgttactctg ttgcagaagg atattattct caccgggaaa ctgtgaacgg ctcatggtac 120 attcaagatt tgtgtgagat gttgggaaaa tatggctcct ccttagagtt cacagaactc 180 ctcacactgg tgaacaggaa agtttctcag cgccgagtgg acttttgcaa agacccaagt 240 gcaattggaa agaagcaggt tccctgtttt gcctcaatgc taactaaaaa gctgcatttc 300 tttccaaaat ctaatctcga gcaccaccac caccaccacg ttgaaattga tgggggatcc 360 cccatgagct cggcctcggg gctccgcagg gggcacccgg caggtgggga agaaaacatg 420 acagaaacag atgccttcta taaaagagaa atgtttgatc cggcagaaaa gtacaaaatg 480 gaccacagga ggagaggaat tgctttaatc ttcaatcatg agaggttctt ttggcactta 540 acactgccag aaaggcgggg cacctgcgca gatagagaca atcttacccg caggttttca 600 gatctaggat ttgaagtgaa atgctttaat gatcttaaag cagaagaact actgctcaaa 660 attcatgagg tgtcaactgt tagccacgca gatgccgatt gctttgtgtg tgtcttcctg 720 agccatggcg aaggcaatca catttatgca tatgatgcta aaatcgaaat tcagacatta 780 actggcttgt tcaaaggaga caagtgtcac agcctggttg gaaaacccaa gatatttatc 840 atccaggcat gtcggggaaa ccagcacgat gtgccagtca ttcctttgga tgtagtagat 900 taa 903 4 1215 DNA Homo sapien 4 atggccgaca aggtcctgaa ggagaagaga aagctgttta tccgttccat gggtgaaggt 60 acaataaatg gcttactgga tgaattatta cagacaaggg tgctgaacaa ggaagagatg 120 gagaaagtaa aacgtgaaaa tgctacagtt atggataaga cccgagcttt gattgactcc 180 gttattccga aaggggcaca ggcatgccaa atttgcatca catacatttg tgaagaagac 240 agttacctgg cagggacgct gggactctca gcagatcaaa catctggaaa ttaccttaat 300 atgcaagact ctcaaggagt actttcttcc tttccagctc ctcaggcagt gcaggacaac 360 ccagctatgc ccacatcctc aggctcagaa gggaatgtca agctttgctc cctagaagaa 420 gctcaaagga tatggaaaca aaagtcggca gagatttatc caataatgga caagtcaagc 480 cgcacacgtc ttgctctcat tatctgcaat gaagaatttg acagtattcc tagaagaact 540 ggagctgagg ttgacatcac aggcatgaca atgctgctac aaaatctggg gtacagcgta 600 gatgtgaaaa aaaatctcac tgcttcggac atgactacag agctggaggc atttgcacac 660 cgcccagagc acaagacctc tgacagcacg ttcctggtgt tcatgtctca tggtattcgg 720 gaaggcattt gtgggaagaa acactctgag caagtcccag atatactaca actcaatgca 780 atctttaaca tgttgaatac caagaactgc ccaagtttga aggacaaacc gaaggtgatc 840 atcatccagg cctgccgtgg tgacagccct ggtgtggtgt ggtttaaaga ttcagtagga 900 gtttctggaa acctatcttt accaactaca gaagagtttg aggatgatgc tattaagaaa 960 gcccacatag agaaggattt tatcgctttc tgctcttcca caccagataa tgtttcttgg 1020 agacatccca caatgggctc tgtttttatt ggaagactca ttgaacatat gcaagaatat 1080 gcctgttcct gtgatgtgga ggaaattttc cgcaaggttc gattttcatt tgagcagcca 1140 gatggtagag cgcagatgcc caccactgaa agagtgactt tgacaagatg tttctacctc 1200 ttcccaggac attaa 1215 5 1215 DNA Homo sapien 5 taccggctgt tccaggactt cctcttctct ttcgacaaat aggcaaggta cccacttcca 60 tgttatttac cgaatgacct acttaataat gtctgttccc acgacttgtt ccttctctac 120 ctctttcatt ttgcactttt acgatgtcaa tacctattct gggctcgaaa ctaactgagg 180 caataaggct ttccccgtgt ccgtacggtt taaacgtagt gtatgtaaac acttcttctg 240 tcaatggacc gtccctgcga ccctgagagt cgtctagttt gtagaccttt aatggaatta 300 tacgttctga gagttcctca tgaaagaagg aaaggtcgag gagtccgtca cgtcctgttg 360 ggtcgatacg ggtgtaggag tccgagtctt cccttacagt tcgaaacgag ggatcttctt 420 cgagtttcct atacctttgt tttcagccgt ctctaaatag gttattacct gttcagttcg 480 gcgtgtgcag aacgagagta atagacgtta cttcttaaac tgtcataagg atcttcttga 540 cctcgactcc aactgtagtg tccgtactgt tacgacgatg ttttagaccc catgtcgcat 600 ctacactttt ttttagagtg acgaagcctg tactgatgtc tcgacctccg taaacgtgtg 660 gcgggtctcg tgttctggag actgtcgtgc aaggaccaca agtacagagt accataagcc 720 cttccgtaaa cacccttctt tgtgagactc gttcagggtc tatatgatgt tgagttacgt 780 tagaaattgt acaacttatg gttcttgacg ggttcaaact tcctgtttgg cttccactag 840 tagtaggtcc ggacggcacc actgtcggga ccacaccaca ccaaatttct aagtcatcct 900 caaagacctt tggatagaaa tggttgatgt cttctcaaac tcctactacg ataattcttt 960 cgggtgtatc tcttcctaaa atagcgaaag acgagaaggt gtggtctatt acaaagaacc 1020 tctgtagggt gttacccgag acaaaaataa ccttctgagt aacttgtata cgttcttata 1080 cggacaagga cactacacct cctttaaaag gcgttccaag ctaaaagtaa actcgtcggt 1140 ctaccatctc gcgtctacgg gtggtgactt tctcactgaa actgttctac aaagatggag 1200 aagggtcctg taatt 1215 6 404 PRT Homo sapien 6 Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile Arg Ser 1 5 10 15 Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr 20 25 30 Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala 35 40 45 Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys 50 55 60 Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp 65 70 75 80 Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly 85 90 95 Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val Leu Ser Ser Phe Pro 100 105 110 Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly 115 120 125 Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile 130 135 140 Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser 145 150 155 160 Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile 165 170 175 Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu 180 185 190 Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205 Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220 Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg 225 230 235 240 Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu 245 250 255 Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser 260 265 270 Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp 275 280 285 Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn 290 295 300 Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys 305 310 315 320 Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp 325 330 335 Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg 340 345 350 Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu 355 360 365 Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala 370 375 380 Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu 385 390 395 400 Phe Pro Gly His 7 1308 DNA Homo sapien 7 atggccgctg acaggggacg caggatattg ggagtgtgtg gcatgcatcc tcatcatcag 60 gaaactctaa aaaagaaccg agtggtgcta gccaaacagc tgttgttgag cgaattgtta 120 gaacatcttc tggagaagga catcatcacc ttggaaatga gggagctcat ccaggccaaa 180 gtgggcagtt tcagccagaa tgtggaactc ctcaacttgc tgcctaagag gggtccccaa 240 gcttttgatg ccttctgtga agcactgagg gagaccaagc aaggccacct ggaggatatg 300 ttgctcacca ccctttctgg gcttcagcat gtactcccac cgttgagctg tgactacgac 360 ttgagtctcc cttttccggt gtgtgagtcc tgtccccttt acaagaagct ccgcctgtcg 420 acagatactg tggaacactc cctagacaat aaagatggtc ctgtctgcct tcaggtgaag 480 ccttgcactc ctgaatttta tcaaacacac ttccagctgg catataggtt gcagtctcgg 540 cctcgtggcc tagcactggt gttgagcaat gtgcacttca ctggagagaa agaactggaa 600 tttcgctctg gaggggatgt ggaccacagt actctagtca ccctcttcaa gcttttgggc 660 tatgacgtcc atgttctatg tgaccagact gcacaggaaa tgcaagagaa actgcagaat 720 tttgcacagt tacctgcaca ccgagtcacg gactcctgca tcgtggcact cctctcgcat 780 ggtgtggagg gcgccatcta tggtgtggat gggaaactgc tccagctcca agaggttttt 840 cagctctttg acaacgccaa ctgcccaagc ctacagaaca aaccaaaaat gttcttcatc 900 caggcctgcc gtggagatga gactgatcgt ggggttgacc aacaagatgg aaagaaccac 960 gcaggatccc ctgggtgcga ggagagtgat gccggtaaag aaaagttgcc gaagatgaga 1020 ctgcccacgc gctcagacat gatatgcggc tatgcctgcc tcaaagggac tgccgccatg 1080 cggaacacca aacgaggttc ctggtacatc gaggctcttg ctcaagtgtt ttctgagcgg 1140 gcttgtgata tgcacgtggc cgacatgctg gttaaggtga acgcacttat caaggatcgg 1200 gaaggttatg ctcctggcac agaattccac cggtgcaagg aaatgtctga atactgcagc 1260 actctgtgcc gccacctcta cctgttccca ggacaccctc ccacatga 1308 8 1308 DNA Homo sapien 8 taccggcgac tgtcccctgc gtcctataac cctcacacac cgtacgtagg agtagtagtc 60 ctttgagatt ttttcttggc tcaccacgat cggtttgtcg acaacaactc gcttaacaat 120 cttgtagaag acctcttcct gtagtagtgg aacctttact ccctcgagta ggtccggttt 180 cacccgtcaa agtcggtctt acaccttgag gagttgaacg acggattctc cccaggggtt 240 cgaaaactac ggaagacact tcgtgactcc ctctggttcg ttccggtgga cctcctatac 300 aacgagtggt gggaaagacc cgaagtcgta catgagggtg gcaactcgac actgatgctg 360 aactcagagg gaaaaggcca cacactcagg acaggggaaa tgttcttcga ggcggacagc 420 tgtctatgac accttgtgag ggatctgtta tttctaccag gacagacgga agtccacttc 480 ggaacgtgag gacttaaaat agtttgtgtg aaggtcgacc gtatatccaa cgtcagagcc 540 ggagcaccgg atcgtgacca caactcgtta cacgtgaagt gacctctctt tcttgacctt 600 aaagcgagac ctcccctaca cctggtgtca tgagatcagt gggagaagtt cgaaaacccg 660 atactgcagg tacaagatac actggtctga cgtgtccttt acgttctctt tgacgtctta 720 aaacgtgtca atggacgtgt ggctcagtgc ctgaggacgt agcaccgtga ggagagcgta 780 ccacacctcc cgcggtagat accacaccta ccctttgacg aggtcgaggt tctccaaaaa 840 gtcgagaaac tgttgcggtt gacgggttcg gatgtcttgt ttggttttta caagaagtag 900 gtccggacgg cacctctact ctgactagca ccccaactgg ttgttctacc tttcttggtg 960 cgtcctaggg gacccacgct cctctcacta cggccatttc ttttcaacgg cttctactct 1020 gacgggtgcg cgagtctgta ctatacgccg atacggacgg agtttccctg acggcggtac 1080 gccttgtggt ttgctccaag gaccatgtag ctccgagaac gagttcacaa aagactcgcc 1140 cgaacactat acgtgcaccg gctgtacgac caattccact tgcgtgaata gttcctagcc 1200 cttccaatac gaggaccgtg tcttaaggtg gccacgttcc tttacagact tatgacgtcg 1260 tgagacacgg cggtggagat ggacaagggt cctgtgggag ggtgtact 1308 9 435 PRT Homo sapien 9 Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met His 1 5 10 15 Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala Lys 20 25 30 Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp Ile 35 40 45 Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser Phe 50 55 60 Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro Gln 65 70 75 80 Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly His 85 90 95 Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val Leu 100 105 110 Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val Cys 115 120 125 Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr Val 130 135 140 Glu His Ser Leu Asp Asn Lys Asp Gly Pro Val Cys Leu Gln Val Lys 145 150 155 160 Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr Arg 165 170 175 Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val His 180 185 190 Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val Asp 195 200 205 His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val His 210 215 220 Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln Asn 225 230 235 240 Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val Ala 245 250 255 Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly Lys 260 265 270 Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Phe Asp Asn Ala Asn Cys 275 280 285 Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Ala Cys Arg 290 295 300 Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gln Asp Gly Lys Asn His 305 310 315 320 Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys Leu 325 330 335 Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr Ala 340 345 350 Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser Trp 355 360 365 Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp Met 370 375 380 His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp Arg 385 390 395 400 Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met Ser 405 410 415 Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly His 420 425 430 Pro Pro Thr 435 10 835 DNA Homo sapien misc_feature (1)...(835) n = A,T,C or G 10 atggagaaca ctgaaaactc agtggattca aaatccatta aaaatttgga accaaagatc 60 atacatggaa gcgaatcaat ggactctgga atatccctgg acaacagtta taaaatggat 120 tatcctgaga tgggtttatg tataataatt aataataaga attttcataa aagcactgga 180 atgacatctc ggtctggtac agatgtcgat gcagcaaacc tcagggaaac attcagaaac 240 ttgaaatatg aagtcaggaa taaaaatgat cttacacgtg aagaaattgt ggaattgatg 300 cgtgatgttt ctaaagaaga tcacagcaaa aggagcagtt ttgtttgtgt gcttctgagc 360 catggtgaag aaggaataat ttttggaaca aatggacctg ttgacctgaa aaaaataaca 420 aactttttca gaggggatcg ttgtagaagt ctaactggaa aacccaaact tttcattatt 480 caggcctgcc gtggtacaga actggactgt ggcattgaga cagacagtgg tgttgatgat 540 gacatggcgt gtcataaaat accagtggat gccgacttct tgtatgcata ctccacagca 600 cctggttatt attcttggcg aaattcaaag gatggctcct ggttcatcca gtcgctttgt 660 gccatgctga aacagtatgc cgacaagctt gaatttatgc acattcttac ccgggttaac 720 cgaaaggtgg caacagaatt tgagtccttt tcctttgacg ctacttttca tgcaaagaaa 780 cagattccat gtattgtttc catgctcaca aaagaactct atttttatca ctaan 835 11 835 DNA Homo sapien misc_feature (1)...(835) n = A,T,C or G 11 tacctcttgt gacttttgag tcacctaagt tttaggtaat ttttaaacct tggtttctag 60 tatgtacctt cgcttagtta cctgagacct tatagggacc tgttgtcaat attttaccta 120 ataggactct acccaaatac atattattaa ttattattct taaaagtatt ttcgtgacct 180 tactgtagag ccagaccatg tctacagcta cgtcgtttgg agtccctttg taagtctttg 240 aactttatac ttcagtcctt atttttacta gaatgtgcac ttctttaaca ccttaactac 300 gcactacaaa gatttcttct agtgtcgttt tcctcgtcaa aacaaacaca cgaagactcg 360 gtaccacttc ttccttatta aaaaccttgt ttacctggac aactggactt tttttattgt 420 ttgaaaaagt ctcccctagc aacatcttca gattgacctt ttgggtttga aaagtaataa 480 gtccggacgg caccatgtct tgacctgaca ccgtaactct gtctgtcacc acaactacta 540 ctgtaccgca cagtatttta tggtcaccta cggctgaaga acatacgtat gaggtgtcgt 600 ggaccaataa taagaaccgc tttaagtttc ctaccgagga ccaagtaggt cagcgaaaca 660 cggtacgact ttgtcatacg gctgttcgaa cttaaatacg tgtaagaatg ggcccaattg 720 gctttccacc gttgtcttaa actcaggaaa aggaaactgc gatgaaaagt acgtttcttt 780 gtctaaggta cataacaaag gtacgagtgt tttcttgaga taaaaatagt gattn 835 12 277 PRT Homo sapien 12 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Asp Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 13 1134 DNA Homo sapien 13 atggcagaag gcaaccacag aaaaaagcca cttaaggtgt tggaatccct gggcaaagat 60 ttcctcactg gtgttttgga taacttggtg gaacaaaatg tactgaactg gaaggaagag 120 gaaaaaaaga aatattacga tgctaaaact gaagacaaag ttcgggtcat ggcagactct 180 atgcaagaga agcaacgtat ggcaggacaa atgcttcttc aaaccttttt taacatagac 240 caaatatccc ccaataaaaa agctcatccg aatatggagg ctggaccacc tgagtcagga 300 gaatctacag atgccctcaa gctttgtcct catgaagaat tcctgagact atgtaaagaa 360 agagctgaag agatctatcc aataaaggag agaaacaacc gcacacgcct ggctctcatc 420 atatgcaata cagagtttga ccatctgcct ccgaggaatg gagctgactt tgacatcaca 480 gggatgaagg agctacttga gggtctggac tatagtgtag atgtagaaga gaatctgaca 540 gccagggata tggagtcagc gctgagggca tttgctacca gaccagagca caagtcctct 600 gacagcacat tcttggtact catgtctcat ggcatcctgg agggaatctg cggaactgtg 660 catgatgaga aaaaaccaga tgtgctgctt tatgacacca tcttccagat attcaacaac 720 cgcaactgcc tcagtctgaa ggacaaaccc aaggtcatca ttgtccaggc ctgcagaggt 780 gcaaaccgtg gggaactgtg ggtcagagac tctccagcat ccttggaagt ggcctcttca 840 cagtcatctg agaacctgga ggaagatgct gtttacaaga cccacgtgga gaaggacttc 900 attgctttct gctcttcaac gccacacaac gtgtcctgga gagacagcac aatgggctct 960 atcttcatca cacaactcat cacatgcttc cagaaatatt cttggtgctg ccacctagag 1020 gaagtatttc ggaaggtaca gcaatcattt gaaactccaa gggccaaagc tcaaatgccc 1080 accatagaac gactgtccat gacaagatat ttctacctct ttcctggcaa ttga 1134 14 1134 DNA Homo sapien 14 taccgtcttc cgttggtgtc ttttttcggt gaattccaca accttaggga cccgtttcta 60 aaggagtgac cacaaaacct attgaaccac cttgttttac atgacttgac cttccttctc 120 ctttttttct ttataatgct acgattttga cttctgtttc aagcccagta ccgtctgaga 180 tacgttctct tcgttgcata ccgtcctgtt tacgaagaag tttggaaaaa attgtatctg 240 gtttataggg ggttattttt tcgagtaggc ttatacctcc gacctggtgg actcagtcct 300 cttagatgtc tacgggagtt cgaaacagga gtacttctta aggactctga tacatttctt 360 tctcgacttc tctagatagg ttatttcctc tctttgttgg cgtgtgcgga ccgagagtag 420 tatacgttat gtctcaaact ggtagacgga ggctccttac ctcgactgaa actgtagtgt 480 ccctacttcc tcgatgaact cccagacctg atatcacatc tacatcttct cttagactgt 540 cggtccctat acctcagtcg cgactcccgt aaacgatggt ctggtctcgt gttcaggaga 600 ctgtcgtgta agaaccatga gtacagagta ccgtaggacc tcccttagac gccttgacac 660 gtactactct tttttggtct acacgacgaa atactgtggt agaaggtcta taagttgttg 720 gcgttgacgg agtcagactt cctgtttggg ttccagtagt aacaggtccg gacgtctcca 780 cgtttggcac cccttgacac ccagtctctg agaggtcgta ggaaccttca ccggagaagt 840 gtcagtagac tcttggacct ccttctacga caaatgttct gggtgcacct cttcctgaag 900 taacgaaaga cgagaagttg cggtgtgttg cacaggacct ctctgtcgtg ttacccgaga 960 tagaagtagt gtgttgagta gtgtacgaag gtctttataa gaaccacgac ggtggatctc 1020 cttcataaag ccttccatgt cgttagtaaa ctttgaggtt cccggtttcg agtttacggg 1080 tggtatcttg ctgacaggta ctgttctata aagatggaga aaggaccgtt aact 1134 15 377 PRT Homo sapien 15 Met Ala Glu Gly Asn His Arg Lys Lys Pro Leu Lys Val Leu Glu Ser 1 5 10 15 Leu Gly Lys Asp Phe Leu Thr Gly Val Leu Asp Asn Leu Val Glu Gln 20 25 30 Asn Val Leu Asn Trp Lys Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala 35 40 45 Lys Thr Glu Asp Lys Val Arg Val Met Ala Asp Ser Met Gln Glu Lys 50 55 60 Gln Arg Met Ala Gly Gln Met Leu Leu Gln Thr Phe Phe Asn Ile Asp 65 70 75 80 Gln Ile Ser Pro Asn Lys Lys Ala His Pro Asn Met Glu Ala Gly Pro 85 90 95 Pro Glu Ser Gly Glu Ser Thr Asp Ala Leu Lys Leu Cys Pro His Glu 100 105 110 Glu Phe Leu Arg Leu Cys Lys Glu Arg Ala Glu Glu Ile Tyr Pro Ile 115 120 125 Lys Glu Arg Asn Asn Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Thr 130 135 140 Glu Phe Asp His Leu Pro Pro Arg Asn Gly Ala Asp Phe Asp Ile Thr 145 150 155 160 Gly Met Lys Glu Leu Leu Glu Gly Leu Asp Tyr Ser Val Asp Val Glu 165 170 175 Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Ala Leu Arg Ala Phe Ala 180 185 190 Thr Arg Pro Glu His Lys Ser Ser Asp Ser Thr Phe Leu Val Leu Met 195 200 205 Ser His Gly Ile Leu Glu Gly Ile Cys Gly Thr Val His Asp Glu Lys 210 215 220 Lys Pro Asp Val Leu Leu Tyr Asp Thr Ile Phe Gln Ile Phe Asn Asn 225 230 235 240 Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Val Gln 245 250 255 Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Trp Val Arg Asp Ser Pro 260 265 270 Ala Ser Leu Glu Val Ala Ser Ser Gln Ser Ser Glu Asn Leu Glu Glu 275 280 285 Asp Ala Val Tyr Lys Thr His Val Glu Lys Asp Phe Ile Ala Phe Cys 290 295 300 Ser Ser Thr Pro His Asn Val Ser Trp Arg Asp Ser Thr Met Gly Ser 305 310 315 320 Ile Phe Ile Thr Gln Leu Ile Thr Cys Phe Gln Lys Tyr Ser Trp Cys 325 330 335 Cys His Leu Glu Glu Val Phe Arg Lys Val Gln Gln Ser Phe Glu Thr 340 345 350 Pro Arg Ala Lys Ala Gln Met Pro Thr Ile Glu Arg Leu Ser Met Thr 355 360 365 Arg Tyr Phe Tyr Leu Phe Pro Gly Asn 370 375 16 1258 DNA Homo sapien misc_feature (1)...(1258) n = A,T,C or G 16 atgttcaaag gtatccttca gagtggattg gataacttcg tgataaacca catgctaaag 60 aacaacgtgg ctggacaaac atctatccag accctagtac ctaatacgga tcaaaagtcg 120 accagtgtaa aaaaagacaa ccacaaaaaa aaaacagtta agatgttgga atacctgggc 180 aaagatgttc ttcatggtgt ttttaattat ttggcaaaac acgatgttct gacattgaag 240 gaagaggaaa agaaaaaata ttatgatgcc aaaattgaag acaaggccct gatcttggta 300 gactctttgc gaaagaatcg cgtggctcat caaatgttta cccaaacact tctcaatatg 360 gaccaaaaga tcaccagtgt aaaacctctt ctgcaaatcg aggctggacc acctgagtca 420 gcagaatcta caaatatact caaactttgt cctcgtgaag aattcctgag actgtgtaaa 480 aaaaatcatg atgagatcta tccaataaaa aagagagagg accgcagacg cctggctctc 540 atcatatgca atacaaagtt tgatcacctg cctgcaagga atggggctca ctatgacatc 600 gtggggatga aaaggctgct tcaaggcctg ggctacactg tggttgacga aaagaatctc 660 acagccaggg atatggagtc agtgctgagg gcatttgctg ccagaccaga gcacaagtcc 720 tctgacagca cgttcttggt actcatgtct catggcatcc tagagggaat ctgcggaact 780 gcgcataaaa agaaaaaacc ggatgtgctg ctttatgaca ccatcttcca gatattcaac 840 aaccgcaact gcctcagtct aaaggacaaa cccaaggtca tcattgtcca ggcctgcaga 900 ggtgaaaaac atggggaact ctgggtcaga gactctccag catccttggc agtcatctct 960 tcacagtcat ctgagaacct ggaggcagat tctgtttgca agatccacga ggagaaggac 1020 ttcattgctt tctgttcttc aacaccacat aacgtgtcct ggagagaccg cacaaggggc 1080 tccatcttca ttacggaact catcacatgc ttccagaaat attcttgctg ctgccaccta 1140 atggaaatat ttcggaaggt acagaaatca tttgaagttc cacaggctaa agcccagatg 1200 cccaccatag aacgagcaac cttgacaaga gatttctacc tctttcctgg caattgan 1258 17 1258 DNA Homo sapien misc_feature (1)...(1258) n = A,T,C or G 17 tacaagtttc cataggaagt ctcacctaac ctattgaagc actatttggt gtacgatttc 60 ttgttgcacc gacctgtttg tagataggtc tgggatcatg gattatgcct agttttcagc 120 tggtcacatt tttttctgtt ggtgtttttt ttttgtcaat tctacaacct tatggacccg 180 tttctacaag aagtaccaca aaaattaata aaccgttttg tgctacaaga ctgtaacttc 240 cttctccttt tcttttttat aatactacgg ttttaacttc tgttccggga ctagaaccat 300 ctgagaaacg ctttcttagc gcaccgagta gtttacaaat gggtttgtga agagttatac 360 ctggttttct agtggtcaca ttttggagaa gacgtttagc tccgacctgg tggactcagt 420 cgtcttagat gtttatatga gtttgaaaca ggagcacttc ttaaggactc tgacacattt 480 tttttagtac tactctagat aggttatttt ttctctctcc tggcgtctgc ggaccgagag 540 tagtatacgt tatgtttcaa actagtggac ggacgttcct taccccgagt gatactgtag 600 cacccctact tttccgacga agttccggac ccgatgtgac accaactgct tttcttagag 660 tgtcggtccc tatacctcag tcacgactcc cgtaaacgac ggtctggtct cgtgttcagg 720 agactgtcgt gcaagaacca tgagtacaga gtaccgtagg atctccctta gacgccttga 780 cgcgtatttt tcttttttgg cctacacgac gaaatactgt ggtagaaggt ctataagttg 840 ttggcgttga cggagtcaga tttcctgttt gggttccagt agtaacaggt ccggacgtct 900 ccactttttg taccccttga gacccagtct ctgagaggtc gtaggaaccg tcagtagaga 960 agtgtcagta gactcttgga cctccgtcta agacaaacgt tctaggtgct cctcttcctg 1020 aagtaacgaa agacaagaag ttgtggtgta ttgcacagga cctctctggc gtgttccccg 1080 aggtagaagt aatgccttga gtagtgtacg aaggtcttta taagaacgac gacggtggat 1140 tacctttata aagccttcca tgtctttagt aaacttcaag gtgtccgatt tcgggtctac 1200 gggtggtatc ttgctcgttg gaactgttct ctaaagatgg agaaaggacc gttaactn 1258 18 418 PRT Homo sapien 18 Met Phe Lys Gly Ile Leu Gln Ser Gly Leu Asp Asn Phe Val Ile Asn 1 5 10 15 His Met Leu Lys Asn Asn Val Ala Gly Gln Thr Ser Ile Gln Thr Leu 20 25 30 Val Pro Asn Thr Asp Gln Lys Ser Thr Ser Val Lys Lys Asp Asn His 35 40 45 Lys Lys Lys Thr Val Lys Met Leu Glu Tyr Leu Gly Lys Asp Val Leu 50 55 60 His Gly Val Phe Asn Tyr Leu Ala Lys His Asp Val Leu Thr Leu Lys 65 70 75 80 Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala Lys Ile Glu Asp Lys Ala 85 90 95 Leu Ile Leu Val Asp Ser Leu Arg Lys Asn Arg Val Ala His Gln Met 100 105 110 Phe Thr Gln Thr Leu Leu Asn Met Asp Gln Lys Ile Thr Ser Val Lys 115 120 125 Pro Leu Leu Gln Ile Glu Ala Gly Pro Pro Glu Ser Ala Glu Ser Thr 130 135 140 Asn Ile Leu Lys Leu Cys Pro Arg Glu Glu Phe Leu Arg Leu Cys Lys 145 150 155 160 Lys Asn His Asp Glu Ile Tyr Pro Ile Lys Lys Arg Glu Asp Arg Arg 165 170 175 Arg Leu Ala Leu Ile Ile Cys Asn Thr Lys Phe Asp His Leu Pro Ala 180 185 190 Arg Asn Gly Ala His Tyr Asp Ile Val Gly Met Lys Arg Leu Leu Gln 195 200 205 Gly Leu Gly Tyr Thr Val Val Asp Glu Lys Asn Leu Thr Ala Arg Asp 210 215 220 Met Glu Ser Val Leu Arg Ala Phe Ala Ala Arg Pro Glu His Lys Ser 225 230 235 240 Ser Asp Ser Thr Phe Leu Val Leu Met Ser His Gly Ile Leu Glu Gly 245 250 255 Ile Cys Gly Thr Ala His Lys Lys Lys Lys Pro Asp Val Leu Leu Tyr 260 265 270 Asp Thr Ile Phe Gln Ile Phe Asn Asn Arg Asn Cys Leu Ser Leu Lys 275 280 285 Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cys Arg Gly Glu Lys His 290 295 300 Gly Glu Leu Trp Val Arg Asp Ser Pro Ala Ser Leu Ala Val Ile Ser 305 310 315 320 Ser Gln Ser Ser Glu Asn Leu Glu Ala Asp Ser Val Cys Lys Ile His 325 330 335 Glu Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro His Asn Val 340 345 350 Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile Phe Ile Thr Glu Leu Ile 355 360 365 Thr Cys Phe Gln Lys Tyr Ser Cys Cys Cys His Leu Met Glu Ile Phe 370 375 380 Arg Lys Val Gln Lys Ser Phe Glu Val Pro Gln Ala Lys Ala Gln Met 385 390 395 400 Pro Thr Ile Glu Arg Ala Thr Leu Thr Arg Asp Phe Tyr Leu Phe Pro 405 410 415 Gly Asn 19 883 DNA Homo sapien misc_feature (1)...(883) n = A,T,C or G 19 atgagctcgg cctcggggct ccgcaggggg cacccggcag gtggggaaga aaacatgaca 60 gaaacagatg ccttctataa aagagaaatg tttgatccgg cagaaaagta caaaatggac 120 cacaggagga gaggaattgc tttaatcttc aatcatgaga ggttcttttg gcacttaaca 180 ctgccagaaa ggcggggcac ctgcgcagat agagacaatc ttacccgcag gttttcagat 240 ctaggatttg aagtgaaatg ctttaatgat cttaaagcag aagaactact gctcaaaatt 300 catgaggtgt caactgttag ccacgcagat gccgattgct ttgtgtgtgt cttcctgagc 360 catggcgaag gcaatcacat ttatgcatat gatgctaaaa tcgaaattca gacattaact 420 ggcttgttca aaggagacaa gtgtcacagc ctggttggaa aacccaagat atttatcatc 480 caggcatgtc ggggaaacca gcacgatgtg ccagtcattc ctttggatgt agtagataat 540 cagacagaga agttggacac caacataact gaggtggatg cagcctccgt ttacacgctg 600 cctgctggag ctgacttcct catgtgttac tctgttgcag aaggatatta ttctcaccgg 660 gaaactgtga acggctcatg gtacattcaa gatttgtgtg agatgttggg aaaatatggc 720 tcctccttag agttcacaga actcctcaca ctggtgaaca ggaaagtttc tcagcgccga 780 gtggactttt gcaaagaccc aagtgcaatt ggaaagaagc aggttccctg ttttgcctca 840 atgctaacta aaaagctgca tttctttcca aaatctaatt aan 883 20 883 DNA Homo sapien misc_feature (1)...(883) n = A,T,C or G 20 tactcgagcc ggagccccga ggcgtccccc gtgggccgtc caccccttct tttgtactgt 60 ctttgtctac ggaagatatt ttctctttac aaactaggcc gtcttttcat gttttacctg 120 gtgtcctcct ctccttaacg aaattagaag ttagtactct ccaagaaaac cgtgaattgt 180 gacggtcttt ccgccccgtg gacgcgtcta tctctgttag aatgggcgtc caaaagtcta 240 gatcctaaac ttcactttac gaaattacta gaatttcgtc ttcttgatga cgagttttaa 300 gtactccaca gttgacaatc ggtgcgtcta cggctaacga aacacacaca gaaggactcg 360 gtaccgcttc cgttagtgta aatacgtata ctacgatttt agctttaagt ctgtaattga 420 ccgaacaagt ttcctctgtt cacagtgtcg gaccaacctt ttgggttcta taaatagtag 480 gtccgtacag cccctttggt cgtgctacac ggtcagtaag gaaacctaca tcatctatta 540 gtctgtctct tcaacctgtg gttgtattga ctccacctac gtcggaggca aatgtgcgac 600 ggacgacctc gactgaagga gtacacaatg agacaacgtc ttcctataat aagagtggcc 660 ctttgacact tgccgagtac catgtaagtt ctaaacacac tctacaaccc ttttataccg 720 aggaggaatc tcaagtgtct tgaggagtgt gaccacttgt cctttcaaag agtcgcggct 780 cacctgaaaa cgtttctggg ttcacgttaa cctttcttcg tccaagggac aaaacggagt 840 tacgattgat ttttcgacgt aaagaaaggt tttagattaa ttn 883 21 293 PRT Homo sapien 21 Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro Ala Gly Gly Glu 1 5 10 15 Glu Asn Met Thr Glu Thr Asp Ala Phe Tyr Lys Arg Glu Met Phe Asp 20 25 30 Pro Ala Glu Lys Tyr Lys Met Asp His Arg Arg Arg Gly Ile Ala Leu 35 40 45 Ile Phe Asn His Glu Arg Phe Phe Trp His Leu Thr Leu Pro Glu Arg 50 55 60 Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Thr Arg Arg Phe Ser Asp 65 70 75 80 Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Leu Lys Ala Glu Glu Leu 85 90 95 Leu Leu Lys Ile His Glu Val Ser Thr Val Ser His Ala Asp Ala Asp 100 105 110 Cys Phe Val Cys Val Phe Leu Ser His Gly Glu Gly Asn His Ile Tyr 115 120 125 Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Leu Thr Gly Leu Phe Lys 130 135 140 Gly Asp Lys Cys His Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Asn Gln His Asp Val Pro Val Ile Pro Leu Asp 165 170 175 Val Val Asp Asn Gln Thr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val 180 185 190 Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gly Ala Asp Phe Leu Met 195 200 205 Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn 210 215 220 Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly 225 230 235 240 Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val 245 250 255 Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys 260 265 270 Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe 275 280 285 Phe Pro Lys Ser Asn 290 22 913 DNA Homo sapien misc_feature (1)...(913) n = A,T,C or G 22 atggcagatg atcagggctg tattgaagag cagggggttg aggattcagc aaatgaagat 60 tcagtggatg ctaagccaga ccggtcctcg tttgtaccgt ccctcttcag taagaagaag 120 aaaaatgtca ccatgcgatc catcaagacc acccgggacc gagtgcctac atatcagtac 180 aacatgaatt ttgaaaagct gggcaaatgc atcataataa acaacaagaa ctttgataaa 240 gtgacaggta tgggcgttcg aaacggaaca gacaaagatg ccgaggcgct cttcaagtgc 300 ttccgaagcc tgggttttga cgtgattgtc tataatgact gctcttgtgc caagatgcaa 360 gatctgctta aaaaagcttc tgaagaggac catacaaatg ccgcctgctt cgcctgcatc 420 ctcttaagcc atggagaaga aaatgtaatt tatgggaaag atggtgtcac accaataaag 480 gatttgacag cccactttag gggggataga tgcaaaaccc ttttagagaa acccaaactc 540 ttcttcattc aggcttgccg agggaccgag cttgatgatg gcatccaggc cgactcgggg 600 cccatcaatg acacagatgc taatcctcga tacaagatcc cagtggaagc tgacttcctc 660 ttcgcctatt ccacggttcc aggctattac tcgtggagga gcccaggaag aggctcctgg 720 tttgtgcaag ccctctgctc catcctggag gagcacggaa aagacctgga aatcatgcag 780 atcctcacca gggtgaatga cagagttgcc aggcactttg agtctcagtc tgatgaccca 840 cacttccatg agaagaagca gatcccctgt gtggtctcca tgctcaccaa ggaactctac 900 ttcagtcaat agn 913 23 913 DNA Homo sapien misc_feature (1)...(913) n = A,T,C or G 23 taccgtctac tagtcccgac ataacttctc gtcccccaac tcctaagtcg tttacttcta 60 agtcacctac gattcggtct ggccaggagc aaacatggca gggagaagtc attcttcttc 120 tttttacagt ggtacgctag gtagttctgg tgggccctgg ctcacggatg tatagtcatg 180 ttgtacttaa aacttttcga cccgtttacg tagtattatt tgttgttctt gaaactattt 240 cactgtccat acccgcaagc tttgccttgt ctgtttctac ggctccgcga gaagttcacg 300 aaggcttcgg acccaaaact gcactaacag atattactga cgagaacacg gttctacgtt 360 ctagacgaat tttttcgaag acttctcctg gtatgtttac ggcggacgaa gcggacgtag 420 gagaattcgg tacctcttct tttacattaa ataccctttc taccacagtg tggttatttc 480 ctaaactgtc gggtgaaatc ccccctatct acgttttggg aaaatctctt tgggtttgag 540 aagaagtaag tccgaacggc tccctggctc gaactactac cgtaggtccg gctgagcccc 600 gggtagttac tgtgtctacg attaggagct atgttctagg gtcaccttcg actgaaggag 660 aagcggataa ggtgccaagg tccgataatg agcacctcct cgggtccttc tccgaggacc 720 aaacacgttc gggagacgag gtaggacctc ctcgtgcctt ttctggacct ttagtacgtc 780 taggagtggt cccacttact gtctcaacgg tccgtgaaac tcagagtcag actactgggt 840 gtgaaggtac tcttcttcgt ctaggggaca caccagaggt acgagtggtt ccttgagatg 900 aagtcagtta tcn 913 24 303 PRT Homo sapien 24 Met Ala Asp Asp Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp 180 185 190 Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn 195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser 210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln 290 295 300 25 1493 DNA Homo sapien misc_feature (1)...(1493) n = A,T,C or G 25 atggacttca gcagaaatct ttatgatatt ggggaacaac tggacagtga agatctggcc 60 tccctcaagt tcctgagcct ggactacatt ccgcaaagga agcaagaacc catcaaggat 120 gccttgatgt tattccagag actccaggaa aagagaatgt tggaggaaag caatctgtcc 180 ttcctgaagg agctgctctt ccgaattaat agactggatt tgctgattac ctacctaaac 240 actagaaagg aggagatgga aagggaactt cagacaccag gcagggctca aatttctgcc 300 tacaggttcc acttctgccg catgagctgg gctgaagcaa acagccagtg ccagacacag 360 tctgtacctt tctggcggag ggtcgatcat ctattaataa gggtcatgct ctatcagatt 420 tcagaagaag tgagcagatc agaattgagg tcttttaagt ttcttttgca agaggaaatc 480 tccaaatgca aactggatga tgacatgaac ctgctggata ttttcataga gatggagaag 540 agggtcatcc tgggagaagg aaagttggac atcctgaaaa gagtctgtgc ccaaatcaac 600 aagagcctgc tgaagataat caacgactat gaagaattca gcaaagggga ggagttgtgt 660 ggggtaatga caatctcgga ctctccaaga gaacaggata gtgaatcaca gactttggac 720 aaagtttacc aaatgaaaag caaacctcgg ggatactgtc tgatcatcaa caatcacaat 780 tttgcaaaag cacgggagaa agtgcccaaa cttcacagca ttagggacag gaatggaaca 840 cacttggatg caggggcttt gaccacgacc tttgaagagc ttcattttga gatcaagccc 900 caccatgact gcacagtaga gcaaatctat gagattttga aaatctacca actcatggac 960 cacagtaaca tggactgctt catctgctgt atcctctccc atggagacaa gggcatcatc 1020 tatggcactg atggacagga ggcccccatc tatgagctga catctcagtt cactggtttg 1080 aagtgccctt cccttgctgg aaaacccaaa gtgtttttta ttcaggcttg tcagggggat 1140 aactaccaga aaggtatacc tgttgagact gattcagagg agcaacccta tttagaaatg 1200 gatttatcat cacctcaaac gagatatatc ccggatgagg ctgactttct gctggggatg 1260 gccactgtga ataactgtgt ttcctaccga aaccctgcag agggaacctg gtacatccag 1320 tcactttgcc agagcctgag agagcgatgt cctcgaggcg atgatattct caccatcctg 1380 actgaagtga actatgaagt aagcaacaag gatgacaaga aaaacatggg gaaacagatg 1440 cctcagccta ctttcacact aagaaaaaaa cttgtcttcc cttctgattg ann 1493 26 1493 DNA Homo sapien misc_feature (1)...(1493) n = A,T,C or G 26 tacctgaagt cgtctttaga aatactataa ccccttgttg acctgtcact tctagaccgg 60 agggagttca aggactcgga cctgatgtaa ggcgtttcct tcgttcttgg gtagttccta 120 cggaactaca ataaggtctc tgaggtcctt ttctcttaca acctcctttc gttagacagg 180 aaggacttcc tcgacgagaa ggcttaatta tctgacctaa acgactaatg gatggatttg 240 tgatctttcc tcctctacct ttcccttgaa gtctgtggtc cgtcccgagt ttaaagacgg 300 atgtccaagg tgaagacggc gtactcgacc cgacttcgtt tgtcggtcac ggtctgtgtc 360 agacatggaa agaccgcctc ccagctagta gataattatt cccagtacga gatagtctaa 420 agtcttcttc actcgtctag tcttaactcc agaaaattca aagaaaacgt tctcctttag 480 aggtttacgt ttgacctact actgtacttg gacgacctat aaaagtatct ctacctcttc 540 tcccagtagg accctcttcc tttcaacctg taggactttt ctcagacacg ggtttagttg 600 ttctcggacg acttctatta gttgctgata cttcttaagt cgtttcccct cctcaacaca 660 ccccattact gttagagcct gagaggttct cttgtcctat cacttagtgt ctgaaacctg 720 tttcaaatgg tttacttttc gtttggagcc cctatgacag actagtagtt gttagtgtta 780 aaacgttttc gtgccctctt tcacgggttt gaagtgtcgt aatccctgtc cttaccttgt 840 gtgaacctac gtccccgaaa ctggtgctgg aaacttctcg aagtaaaact ctagttcggg 900 gtggtactga cgtgtcatct cgtttagata ctctaaaact tttagatggt tgagtacctg 960 gtgtcattgt acctgacgaa gtagacgaca taggagaggg tacctctgtt cccgtagtag 1020 ataccgtgac tacctgtcct ccgggggtag atactcgact gtagagtcaa gtgaccaaac 1080 ttcacgggaa gggaacgacc ttttgggttt cacaaaaaat aagtccgaac agtcccccta 1140 ttgatggtct ttccatatgg acaactctga ctaagtctcc tcgttgggat aaatctttac 1200 ctaaatagta gtggagtttg ctctatatag ggcctactcc gactgaaaga cgacccctac 1260 cggtgacact tattgacaca aaggatggct ttgggacgtc tcccttggac catgtaggtc 1320 agtgaaacgg tctcggactc tctcgctaca ggagctccgc tactataaga gtggtaggac 1380 tgacttcact tgatacttca ttcgttgttc ctactgttct ttttgtaccc ctttgtctac 1440 ggagtcggat gaaagtgtga ttcttttttt gaacagaagg gaagactaac tnn 1493 27 476 PRT Homo sapien 27 Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser 1 5 10 15 Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln 20 25 30 Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu 35 40 45 Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu 50 55 60 Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn 65 70 75 80 Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala 85 90 95 Gln Ile Ser Ala Tyr Arg Phe His Phe Cys Arg Met Ser Trp Ala Glu 100 105 110 Ala Asn Ser Gln Cys Gln Thr Gln Ser Val Pro Phe Trp Arg Arg Val 115 120 125 Asp His Leu Leu Ile Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val 130 135 140 Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile 145 150 155 160 Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile 165 170 175 Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu 180 185 190 Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn 195 200 205 Asp Tyr Glu Glu Phe Ser Lys Gly Glu Glu Leu Cys Lys Val Tyr Gln 210 215 220 Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn Asn His Asn 225 230 235 240 Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His Ser Ile Arg Asp 245 250 255 Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr Thr Phe Glu 260 265 270 Glu Leu His Phe Glu Ile Lys Pro His His Asp Cys Thr Val Glu Gln 275 280 285 Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu Met Asp His Ser Asn Met 290 295 300 Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gly Asp Lys Gly Ile Ile 305 310 315 320 Tyr Gly Thr Asp Gly Gln Glu Ala Pro Ile Tyr Glu Leu Thr Ser Gln 325 330 335 Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro Lys Val Phe 340 345 350 Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly Ile Pro Val 355 360 365 Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met Asp Leu Ser Ser 370 375 380 Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe Leu Leu Gly Met 385 390 395 400 Ala Thr Val Asn Asn Cys Val Ser Tyr Arg Asn Pro Ala Glu Gly Thr 405 410 415 Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu Arg Glu Arg Cys Pro Arg 420 425 430 Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr Glu Val Ser 435 440 445 Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gln Met Pro Gln Pro Thr 450 455 460 Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Ser Asp 465 470 475 28 1252 DNA Homo sapien misc_feature (1)...(1252) n = A,T,C or G 28 atggacgaag cggatcggcg gctcctgcgg cggtgccggc tgcggctggt ggaagagctg 60 caggtggacc agctctggga cgccctgctg agcagcgagc tgttcaggcc ccatatgatc 120 gaggacatcc agcgggcagg ctctggatct cggcgggatc aggccaggca gctgatcata 180 gatctggaga ctcgagggag tcaggctctt cctttgttca tctcctgctt agaggacaca 240 ggccaggaca tgctggcttc gtttctgcga actaacaggc aagcagcaaa gttgtcgaag 300 ccaaccctag aaaaccttac cccagtggtg ctcagaccag agattcgcaa accagaggtt 360 ctcagaccgg aaacacccag accagtggac attggttctg gaggatttgg tgatgtcggt 420 gctcttgaga gtttgagggg aaatgcagat ttggcttaca tcctgagcat ggagccctgt 480 ggccactgcc tcattatcaa caatgtgaac ttctgccgtg agtccgggct ccgcacccgc 540 actggctcca acatcgactg tgagaagttg cggcgtcgct tctcctcgcc gcatttcatg 600 gtggaggtga agggcgacct gactgccaag aaaatggtgc tggctttgct ggagctggcg 660 cggcaggacc acggtgctct ggactgctgc gtggtggtca ttctctctca cggctgtcag 720 gccagccacc tgcagttccc aggggctgtc tacggcacag atggatgccc tgtgtcggtc 780 gagaagattg tgaacatctt caatgggacc agctgcccca gcctgggagg aaagcccaag 840 ctctttttca tccaggcctg tggtggggag cagaaagacc atgggtttga ggtggcctcc 900 acttcccctg aagacgagtc ccctggcagt aaccccgagc cagatgccac cccgttccag 960 gaaggtttga ggaccttcga ccagctggac gccatatcta gtttgcccac acccagtgac 1020 atctttgtgt cctactctac tttcccaggt tttgtttcct ggagggaccc caagagtggc 1080 tcctggtacg ttgagaccct ggacgacatc tttgagcagt gggctcactc tgaagacctg 1140 cagtccctcc tgcttagggt cgctaatgct gtttcggtga aagggattta taaacagatg 1200 cctggttgct ttaatttcct ccggaaaaaa cttttcttta aaacatcata an 1252 29 1252 DNA Homo sapien misc_feature (1)...(1252) n = A,T,C or G 29 tacctgcttc gcctagccgc cgaggacgcc gccacggccg acgccgacca ccttctcgac 60 gtccacctgg tcgagaccct gcgggacgac tcgtcgctcg acaagtccgg ggtatactag 120 ctcctgtagg tcgcccgtcc gagacctaga gccgccctag tccggtccgt cgactagtat 180 ctagacctct gagctccctc agtccgagaa ggaaacaagt agaggacgaa tctcctgtgt 240 ccggtcctgt acgaccgaag caaagacgct tgattgtccg ttcgtcgttt caacagcttc 300 ggttgggatc ttttggaatg gggtcaccac gagtctggtc tctaagcgtt tggtctccaa 360 gagtctggcc tttgtgggtc tggtcacctg taaccaagac ctcctaaacc actacagcca 420 cgagaactct caaactcccc tttacgtcta aaccgaatgt aggactcgta cctcgggaca 480 ccggtgacgg agtaatagtt gttacacttg aagacggcac tcaggcccga ggcgtgggcg 540 tgaccgaggt tgtagctgac actcttcaac gccgcagcga agaggagcgg cgtaaagtac 600 cacctccact tcccgctgga ctgacggttc ttttaccacg accgaaacga cctcgaccgc 660 gccgtcctgg tgccacgaga cctgacgacg caccaccagt aagagagagt gccgacagtc 720 cggtcggtgg acgtcaaggg tccccgacag atgccgtgtc tacctacggg acacagccag 780 ctcttctaac acttgtagaa gttaccctgg tcgacggggt cggaccctcc tttcgggttc 840 gagaaaaagt aggtccggac accacccctc gtctttctgg tacccaaact ccaccggagg 900 tgaaggggac ttctgctcag gggaccgtca ttggggctcg gtctacggtg gggcaaggtc 960 cttccaaact cctggaagct ggtcgacctg cggtatagat caaacgggtg tgggtcactg 1020 tagaaacaca ggatgagatg aaagggtcca aaacaaagga cctccctggg gttctcaccg 1080 aggaccatgc aactctggga cctgctgtag aaactcgtca cccgagtgag acttctggac 1140 gtcagggagg acgaatccca gcgattacga caaagccact ttccctaaat atttgtctac 1200 ggaccaacga aattaaagga ggcctttttt gaaaagaaat tttgtagtat tn 1252 30 416 PRT Homo sapien 30 Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu 1 5 10 15 Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Ser 20 25 30 Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser 35 40 45 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr 50 55 60 Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr 65 70 75 80 Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala 85 90 95 Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg 100 105 110 Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro 115 120 125 Val Asp Ile Gly Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser 130 135 140 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys 145 150 155 160 Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly 165 170 175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg 180 185 190 Arg Phe Ser Ser Pro His Phe Met Val Glu Val Lys Gly Asp Leu Thr 195 200 205 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Arg Gln Asp His 210 215 220 Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln 225 230 235 240 Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys 245 250 255 Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys 260 265 270 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly 275 280 285 Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu 290 295 300 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln 305 310 315 320 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro 325 330 335 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val 340 345 350 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp 355 360 365 Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 370 375 380 Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385 390 395 400 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 405 410 415 31 1440 DNA Homo sapien 31 atgaaatctc aaggtcaaca ttggtattcc agttcagata aaaactgtaa agtgagcttt 60 cgtgagaagc ttctgattat tgattcaaac ctgggggtcc aagatgtgga gaacctcaag 120 tttctctgca taggattggt ccccaacaag aagctggaga agtccagctc agcctcagat 180 gtttttgaac atctcttggc agaggatctg ctgagtgagg aagacccttt cttcctggca 240 gaactcctct atatcatacg gcagaagaag ctgctgcagc acctcaactg taccaaagag 300 gaagtggagc gactgctgcc cacccgacaa agggtttctc tgtttagaaa cctgctctac 360 gaactgtcag aaggcattga ctcagagaac ttaaaggaca tgatcttcct tctgaaagac 420 tcgcttccca aaactgaaat gacctcccta agtttcctgg catttctaga gaaacaaggt 480 aaaatagatg aagataatct gacatgcctg gaggacctct gcaaaacagt tgtacctaaa 540 cttttgagaa acatagagaa atacaaaaga gagaaagcta tccagatagt gacacctcct 600 gtagacaagg aagccgagtc gtatcaagga gaggaagaac tagtttccca aacagatgtt 660 aagacattct tggaagcctt accgagggca gctgtgtaca ggatgaatcg gaaccacaga 720 ggcctctgtg tcattgtcaa caaccacagc tttacctccc tgaaggacag acaaggaacc 780 cataaagatg ctgagatcct gagtcatgtg ttccagtggc ttgggttcac agtgcatata 840 cacaataatg tgacgaaagt ggaaatggag atggtcctgc agaagcagaa gtgcaatcca 900 gcccatgccg acggggactg cttcgtgttc tgtattctga cccatgggag atttggagct 960 gtctactctt cggatgaggc cctcattccc attcgggaga tcatgtctca cttcacagcc 1020 ctgcagtgcc ctagactggc tgaaaaacct aaactctttt tcatccaggc ctgccaaggt 1080 gaagagatac agccttccgt atccatcgaa gcagatgctc tgaaccctga gcaggcaccc 1140 acttccctgc aggacagtat tcctgccgag gctgacttcc tacttggtct ggccactgtc 1200 ccaggctatg tatcctttcg gcatgtggag gaaggcagct ggtatattca gtctctgtgt 1260 aatcatctga agaaattggt cccaagacat gaagacatct tatccatcct cactgctgtc 1320 aacgatgatg tgagtcgaag agtggacaaa cagggaacaa agaaacagat gccccagcct 1380 gctttcacac taaggaaaaa actagtattc cctgtgcccc tggatgcact ttcaatatag 1440 32 1440 DNA Homo sapien 32 tactttagag ttccagttgt aaccataagg tcaagtctat ttttgacatt tcactcgaaa 60 gcactcttcg aagactaata actaagtttg gacccccagg ttctacacct cttggagttc 120 aaagagacgt atcctaacca ggggttgttc ttcgacctct tcaggtcgag tcggagtcta 180 caaaaacttg tagagaaccg tctcctagac gactcactcc ttctgggaaa gaaggaccgt 240 cttgaggaga tatagtatgc cgtcttcttc gacgacgtcg tggagttgac atggtttctc 300 cttcacctcg ctgacgacgg gtgggctgtt tcccaaagag acaaatcttt ggacgagatg 360 cttgacagtc ttccgtaact gagtctcttg aatttcctgt actagaagga agactttctg 420 agcgaagggt tttgacttta ctggagggat tcaaaggacc gtaaagatct ctttgttcca 480 ttttatctac ttctattaga ctgtacggac ctcctggaga cgttttgtca acatggattt 540 gaaaactctt tgtatctctt tatgttttct ctctttcgat aggtctatca ctgtggagga 600 catctgttcc ttcggctcag catagttcct ctccttcttg atcaaagggt ttgtctacaa 660 ttctgtaaga accttcggaa tggctcccgt cgacacatgt cctacttagc cttggtgtct 720 ccggagacac agtaacagtt gttggtgtcg aaatggaggg acttcctgtc tgttccttgg 780 gtatttctac gactctagga ctcagtacac aaggtcaccg aacccaagtg tcacgtatat 840 gtgttattac actgctttca cctttacctc taccaggacg tcttcgtctt cacgttaggt 900 cgggtacggc tgcccctgac gaagcacaag acataagact gggtaccctc taaacctcga 960 cagatgagaa gcctactccg ggagtaaggg taagccctct agtacagagt gaagtgtcgg 1020 gacgtcacgg gatctgaccg actttttgga tttgagaaaa agtaggtccg gacggttcca 1080 cttctctatg tcggaaggca taggtagctt cgtctacgag acttgggact cgtccgtggg 1140 tgaagggacg tcctgtcata aggacggctc cgactgaagg atgaaccaga ccggtgacag 1200 ggtccgatac ataggaaagc cgtacacctc cttccgtcga ccatataagt cagagacaca 1260 ttagtagact tctttaacca gggttctgta cttctgtaga ataggtagga gtgacgacag 1320 ttgctactac actcagcttc tcacctgttt gtcccttgtt tctttgtcta cggggtcgga 1380 cgaaagtgtg attccttttt tgatcataag ggacacgggg acctacgtga aagttatatc 1440 33 479 PRT Homo sapien 33 Met Lys Ser Gln Gly Gln His Trp Tyr Ser Ser Ser Asp Lys Asn Cys 1 5 10 15 Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Ile Asp Ser Asn Leu Gly 20 25 30 Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cys Ile Gly Leu Val Pro 35 40 45 Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Ser Asp Val Phe Glu His 50 55 60 Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu Asp Pro Phe Phe Leu Ala 65 70 75 80 Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Leu Leu Gln His Leu Asn 85 90 95 Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pro Thr Arg Gln Arg Val 100 105 110 Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Ser Glu Gly Ile Asp Ser 115 120 125 Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Lys Asp Ser Leu Pro Lys 130 135 140 Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Phe Leu Glu Lys Gln Gly 145 150 155 160 Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Glu Asp Leu Cys Lys Thr 165 170 175 Val Val Pro Lys Leu Leu Arg Asn Ile Glu Lys Tyr Lys Arg Glu Lys 180 185 190 Ala Ile Gln Ile Val Thr Pro Pro Val Asp Lys Glu Ala Glu Ser Tyr 195 200 205 Gln Gly Glu Glu Glu Leu Val Ser Gln Thr Asp Val Lys Thr Phe Leu 210 215 220 Glu Ala Leu Pro Arg Ala Ala Val Tyr Arg Met Asn Arg Asn His Arg 225 230 235 240 Gly Leu Cys Val Ile Val Asn Asn His Ser Phe Thr Ser Leu Lys Asp 245 250 255 Arg Gln Gly Thr His Lys Asp Ala Glu Ile Leu Ser His Val Phe Gln 260 265 270 Trp Leu Gly Phe Thr Val His Ile His Asn Asn Val Thr Lys Val Glu 275 280 285 Met Glu Met Val Leu Gln Lys Gln Lys Cys Asn Pro Ala His Ala Asp 290 295 300 Gly Asp Cys Phe Val Phe Cys Ile Leu Thr His Gly Arg Phe Gly Ala 305 310 315 320 Val Tyr Ser Ser Asp Glu Ala Leu Ile Pro Ile Arg Glu Ile Met Ser 325 330 335 His Phe Thr Ala Leu Gln Cys Pro Arg Leu Ala Glu Lys Pro Lys Leu 340 345 350 Phe Phe Ile Gln Ala Cys Gln Gly Glu Glu Ile Gln Pro Ser Val Ser 355 360 365 Ile Glu Ala Asp Ala Leu Asn Pro Glu Gln Ala Pro Thr Ser Leu Gln 370 375 380 Asp Ser Ile Pro Ala Glu Ala Asp Phe Leu Leu Gly Leu Ala Thr Val 385 390 395 400 Pro Gly Tyr Val Ser Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile 405 410 415 Gln Ser Leu Cys Asn His Leu Lys Lys Leu Val Pro Arg His Glu Asp 420 425 430 Ile Leu Ser Ile Leu Thr Ala Val Asn Asp Asp Val Ser Arg Arg Val 435 440 445 Asp Lys Gln Gly Thr Lys Lys Gln Met Pro Gln Pro Ala Phe Thr Leu 450 455 460 Arg Lys Lys Leu Val Phe Pro Val Pro Leu Asp Ala Leu Ser Ile 465 470 475 34 290 PRT Artificial Sequence Rev-Caspase-3 contructed from human caspase -3 34 Met Ile Glu Thr Asp Ser Gly Val Asp Asp Asp Met Ala Cys His Lys 1 5 10 15 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly 20 25 30 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser 35 40 45 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 50 55 60 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe 65 70 75 80 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val 85 90 95 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His Asp Glu Val Asp Gly 100 105 110 Gly Ser Pro Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile 115 120 125 Lys Asn Leu Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser 130 135 140 Gly Ile Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly 145 150 155 160 Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met 165 170 175 Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr 180 185 190 Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg 195 200 205 Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser 210 215 220 Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly 225 230 235 240 Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn 245 250 255 Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu 260 265 270 Phe Ile Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu 275 280 285 Thr Asp 290 35 285 PRT Artificial Sequence Uncleavable Rev-Caspase-3 contructed from human caspase -3 35 Met Ile Glu Thr Asp Ser Gly Val Asp Asp Asp Met Ala Cys His Lys 1 5 10 15 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly 20 25 30 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser 35 40 45 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 50 55 60 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe 65 70 75 80 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val 85 90 95 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His Gly Ser Pro Met Glu 100 105 110 Asn Thr Glu Asn Ser Val Ala Ser Lys Ser Ile Lys Asn Leu Glu Pro 115 120 125 Lys Ile Ile His Gly Ser Glu Ser Met Ala Ser Gly Ile Ser Leu Asp 130 135 140 Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile Ile Ile 145 150 155 160 Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg Ser Gly 165 170 175 Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn Leu Lys 180 185 190 Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile Val Glu 195 200 205 Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser Ser Phe 210 215 220 Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe Gly Thr 225 230 235 240 Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg Gly Asp 245 250 255 Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile Gln Ala 260 265 270 Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp 275 280 285 36 300 PRT Artificial Sequence Rev-Caspase-6 contructed from human caspase-6 36 Met Val Glu Ile Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gly Ala 1 5 10 15 Asp Phe Leu Met Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg 20 25 30 Glu Thr Val Asn Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu 35 40 45 Gly Lys Tyr Gly Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val 50 55 60 Asn Arg Lys Val Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser 65 70 75 80 Ala Ile Gly Lys Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys 85 90 95 Lys Leu His Phe Phe Pro Lys Ser Asn Leu Glu His His His His His 100 105 110 His Val Glu Ile Asp Gly Gly Ser Pro Met Ser Ser Ala Ser Gly Leu 115 120 125 Arg Arg Gly His Pro Ala Gly Gly Glu Glu Asn Met Thr Glu Thr Asp 130 135 140 Ala Phe Tyr Lys Arg Glu Met Phe Asp Pro Ala Glu Lys Tyr Lys Met 145 150 155 160 Asp His Arg Arg Arg Gly Ile Ala Leu Ile Phe Asn His Glu Arg Phe 165 170 175 Phe Trp His Leu Thr Leu Pro Glu Arg Arg Gly Thr Cys Ala Asp Arg 180 185 190 Asp Asn Leu Thr Arg Arg Phe Ser Asp Leu Gly Phe Glu Val Lys Cys 195 200 205 Phe Asn Asp Leu Lys Ala Glu Glu Leu Leu Leu Lys Ile His Glu Val 210 215 220 Ser Thr Val Ser His Ala Asp Ala Asp Cys Phe Val Cys Val Phe Leu 225 230 235 240 Ser His Gly Glu Gly Asn His Ile Tyr Ala Tyr Asp Ala Lys Ile Glu 245 250 255 Ile Gln Thr Leu Thr Gly Leu Phe Lys Gly Asp Lys Cys His Ser Leu 260 265 270 Val Gly Lys Pro Lys Ile Phe Ile Ile Gln Ala Cys Arg Gly Asn Gln 275 280 285 His Asp Val Pro Val Ile Pro Leu Asp Val Val Asp 290 295 300 37 5 PRT Artificial Sequence Protease sensitive linker region between the small and large subunits of a Rev-caspase 37 Asp Xaa Xaa Asp Gly 1 5 38 5 PRT Artificial Sequence Protease sensitive linker region between the small and large subunits of a Rev-caspase 38 Asp Glu Val Asp Gly 1 5 39 5 PRT Artificial Sequence Protease sensitive linker region between the small and large subunits of a Rev-caspase 39 Ile Glu Thr Asp Gly 1 5 40 5 PRT Artificial Sequence Protease sensitive linker region between the small and large subunits of a Rev-caspase 40 Tyr Val Ala Asp Gly 1 5 41 5 PRT Artificial Sequence Protease sensitive linker region between the small and large subunits of a Rev-caspase 41 Tyr Val His Asp Gly 1 5 42 5 PRT Artificial Sequence Protease sensitive linker region between the small and large subunits of a Rev-caspase 42 Trp Glu His Asp Gly 1 5 43 22 DNA Artificial Sequence PCR primer for large subunit of caspase-3 43 atggagaaca ctgaaaactc ag 22 44 22 DNA Artificial Sequence PCR primer for large subunit of caspase-3 44 gtcatcatca acacctcagt ct 22 45 26 DNA Artificial Sequence PCR primer for small subunit of caspase-3 45 ggatccatga ttgagacaga cagtgg 26 46 30 DNA Artificial Sequence PCR primer for small subunit of caspase-3 46 atcaacttca tcgtgataaa aatagagttc 30 47 18 DNA Artificial Sequence PCR primer for large subunit of caspase-6 47 atgagctcgg cctcgggg 18 48 21 DNA Artificial Sequence PCR primer for large subunit of caspase-6 48 ttaatctact acatccaaag g 21 49 36 DNA Artificial Sequence PCR primer for small subunit of caspase-6 49 ggatccatgg tagaaataga tgcagcctcc gtttac 36 50 30 DNA Artificial Sequence PCR primer for small subunit of caspase-6 50 atcaattcaa cgtggtggtg gtggtggtgc 30 51 5 PRT Homo sapien VARIANT (1)...(5) Xaa = Any Amino Acid 51 Gln Ala Cys Xaa Gly 1 5 52 4 PRT Homo Sapien 52 Asp Glu Val Asp 1 53 4 PRT Homo sapien 53 Ile Glu Thr Asp 1 54 6 PRT Homo sapien 54 Arg Thr Arg Thr Gly Ser 1 5 55 6 PRT Homo sapien 55 Leu Ser His Gly Cys Gln 1 5 56 9 PRT Homo sapien 56 Phe Ile Gln Ala Cys Gly Gly Glu Gln 1 5 57 5 PRT Homo sapien 57 Pro Glu Pro Asp Ala 1 5 58 5 PRT Homo sapien 58 Asp Gln Leu Asp Ala 1 5 59 15 PRT Homo sapien 59 Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val 1 5 10 15 60 6 PRT Homo sapien 60 Arg Asp Arg Asn Gly Thr 1 5 61 6 PRT Homo sapien 61 Leu Ser His Gly Asp Lys 1 5 62 9 PRT Homo sapien 62 Phe Ile Gln Ala Cys Gln Gly Asp Asn 1 5 63 5 PRT Homo sapien 63 Val Glu Thr Asp Ser 1 5 64 5 PRT Homo sapien 64 Leu Glu Met Asp Leu 1 5 65 15 PRT Homo sapien 65 Asn Cys Val Ser Tyr Arg Asn Pro Ala Glu Gly Thr Trp Tyr Ile 1 5 10 15 66 6 PRT Homo sapien 66 Lys Asp Arg Gln Gly Thr 1 5 67 6 PRT Homo sapien 67 Leu Thr His Gly Arg Phe 1 5 68 9 PRT Homo sapien 68 Phe Ile Gln Ala Cys Gln Gly Glu Glu 1 5 69 5 PRT Homo sapien 69 Ile Glu Ala Asp Ala 1 5 70 15 PRT Homo sapien 70 Gly Tyr Val Ser Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile 1 5 10 15 71 6 PRT Homo sapien 71 Gly Val Arg Asn Gly Thr 1 5 72 6 PRT Homo sapien 72 Leu Ser His Gly Glu Glu 1 5 73 9 PRT Homo sapien 73 Phe Ile Gln Ala Cys Arg Gly Thr Glu 1 5 74 5 PRT Homo sapien 74 Ile Gln Ala Asp Ser 1 5 75 15 PRT Homo sapien 75 Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp Phe Val 1 5 10 15 76 6 PRT Homo sapien 76 Pro Glu Arg Arg Gly Thr 1 5 77 6 PRT Homo sapien 77 Leu Ser His Gly Glu Gly 1 5 78 9 PRT Homo sapien 78 Ile Ile Gln Ala Cys Arg Gly Asn Gln 1 5 79 5 PRT Homo sapien 79 Asp Val Val Asp Asn 1 5 80 5 PRT Homo sapien 80 Thr Glu Val Asp Ala 1 5 81 15 PRT Homo sapien 81 Gly Tyr Tyr Ser His Arg Glu Thr Val Asn Gly Ser Trp Tyr Ile 1 5 10 15 82 6 PRT Homo sapien 82 Thr Ser Arg Ser Gly Thr 1 5 83 6 PRT Homo sapien 83 Leu Ser His Gly Glu Glu 1 5 84 9 PRT Homo sapien 84 Ile Ile Gln Ala Cys Arg Gly Thr Glu 1 5 85 5 PRT Homo sapien 85 Ile Glu Thr Asp Ser 1 5 86 15 PRT Homo sapien 86 Gly Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile 1 5 10 15 87 6 PRT Homo sapien 87 Pro Thr Arg Asn Gly Thr 1 5 88 6 PRT Homo sapien 88 Leu Ser His Gly Glu Glu 1 5 89 9 PRT Homo sapien 89 Phe Val Gln Ala Cys Arg Gly Glu Arg 1 5 90 5 PRT Homo sapien 90 Asp Ser Val Asp Gly 1 5 91 15 PRT Homo sapien 91 Gln Tyr Val Ser Trp Arg Asn Ser Ala Arg Gly Ser Trp Phe Ile 1 5 10 15 92 6 PRT Homo sapien 92 Pro Arg Arg Thr Gly Ala 1 5 93 6 PRT Homo sapien 93 Met Ser His Gly Ile Arg 1 5 94 9 PRT Homo sapien 94 Ile Ile Gln Ala Cys Arg Gly Asp Ser 1 5 95 5 PRT Homo sapien 95 Trp Phe Lys Asp Ser 1 5 96 5 PRT Homo sapien 96 Phe Glu Asp Asp Ala 1 5 97 15 PRT Homo sapien 97 Asp Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile 1 5 10 15 98 6 PRT Homo sapien 98 Pro Pro Arg Asn Gly Ala 1 5 99 6 PRT Homo sapien 99 Met Ser His Gly Ile Leu 1 5 100 9 PRT Homo sapien 100 Ile Val Gln Ala Cys Arg Gly Ala Asn 1 5 101 5 PRT Homo sapien 101 Trp Val Lys Asp Ser 1 5 102 5 PRT Homo sapien 102 Leu Glu Glu Asp Ala 1 5 103 15 PRT Homo sapien 103 His Asn Val Ser Trp Arg Asp Ser Thr Met Gly Ser Ile Phe Ile 1 5 10 15 104 6 PRT Homo sapien 104 Pro Ala Arg Asn Gly Ala 1 5 105 6 PRT Homo sapien 105 Met Ser His Gly Ile Leu 1 5 106 9 PRT Homo sapien 106 Ile Val Gln Ala Cys Arg Gly Glu Lys 1 5 107 5 PRT Homo sapien 107 Trp Val Arg Asp Ser 1 5 108 5 PRT Homo sapien 108 Leu Glu Ala Asp Ser 1 5 109 15 PRT Homo sapien 109 His Asn Val Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile Phe Ile 1 5 10 15 110 6 PRT Homo sapien 110 Glu Phe Arg Ser Gly Gly 1 5 111 6 PRT Homo sapien 111 Leu Ser His Gly Val Glu 1 5 112 9 PRT Homo sapien 112 Phe Ile Gln Ala Cys Arg Gly Asp Glu 1 5 113 5 PRT Homo sapien 113 Asp Gln Gln Asp Gly 1 5 114 5 PRT Homo sapien 114 Glu Glu Ser Asp Ala 1 5 115 15 PRT Homo sapien 115 Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser Trp Tyr Ile 1 5 10 15 116 5 PRT Artificial Sequence Protease sensitive linker region between the small and large subunits of a Rev-caspase 116 Val Glu Ile Asp Ser 1 5 

We claim:
 1. A method of identifying an inhibitor or enhancer of caspase processing activity, comprising: (a) contacting a sample containing an in vitro translated rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.
 2. A method of identifying an inhibitor or enhancer of caspase processing activity, comprising: (a) contacting a cell transfected with a vector expressing rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.
 3. A method of identifying an inhibitor or enhancer of caspase-mediated apoptosis, comprising: (a) contacting a cell transfected with a vector expressing rev-caspase with a candidate inhibitor or candidate enhancer or with a reference compound; and (b) detecting cell viability, wherein viability of cells contacted with a candidate is increased in the presence of an inhibitor and is decreased in the presence of an enhancer compared to cells contacted with a reference compound.
 4. The method of any one of claims 1-3, wherein the rev-caspase is selected from the group consisting of rev-caspase-1, rev-caspase-2, rev-caspase-3, rev-caspase-4, rev-caspase-5, rev-caspase-6, rev-caspase-7, rev-caspase-8, rev-caspase-9, rev-caspase-10, rev-caspase-11, rev-caspase-12, rev-caspase-13, and rev-caspase-14.
 5. The method of either of claims 1 or 2, wherein detecting comprises gel electrophoresis.
 6. The method of claim 1, wherein the in vitro translated rev-caspase is labeled.
 7. The method of claim 6, wherein the label is a radioactive label, a peptide tag, an enzyme or biotin. 